Novel potassium channels and genes encoding these potassium channels

ABSTRACT

This invention relates to novel potassium channels and genes encoding these channels. More specifically the invention provides isolated polynucleotides encoding the KCNQ5 potassium channel subunit, cells transformed with these polynucleotides, transgenic animals comprising genetic mutations, and the use of the transformed cells and the transgenic animals for the in vitro and in vivo screening of chemical compounds affecting KCNQ5 subunit containing potassium channels.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of co-pending application Ser.No. 09/590,304, filed on Jun. 9, 2000 (for which priority is claimedunder 35 U.S.C. §120), which is a conversion of provisional applicationU.S. 60/139,891 filed Jun. 22, 1999 (for which priority is claimed under35 U.S.C. §119). The entire contents of both of these applications arehereby incorporated by reference. This application also claims priorityof Application No. PA1999-00828 filed in Denmark on Jun. 11, 1999 under35 U.S.C. § 119.

TECHNICAL FIELD

[0002] This invention relates to novel potassium channels and genesencoding these channels. More specifically the invention providesisolated polynucleotides encoding the KCNQ5 potassium channel subunit,cells transformed with these polynucleotides, transgenic animalscomprising genetic mutations, and the use of the transformed cells andthe transgenic animals for the in vitro and in vivo screening ofchemical compounds affecting KCNQ5 subunit containing potassiumchannels.

BACKGROUND ART

[0003] Potassium channels participate in the regulation of electricalsignalling in excitable cells, and regulates the ionic composition ofbiological fluids. Mutations in the four known genes of the KCNQ branchof the K⁺-channel gene family underlie inherited cardiac arrhythmia's,in some cases associated with deafness, neonatal epilepsy, and theprogressive hearing loss of the elderly (presbyacusis).

[0004] Ion channels play important roles in signal transduction and inthe regulation of the ionic composition of intra- and extracellularfluids. KCNO1 is a typical member of the voltage-gated potassium channelsuperfamily with 6 transmembrane domains and a pore region situatedbetween the fifth and the sixth transmembrane domain. The minK protein(also known as KCNE1 or IsK) has a single transmembrane span and cannotform potassium channels on its own. However, as a β-subunit it enhancesand modifies currents mediated by KCNQ1. These heteromeric channelsparticipate in the repolarization of the heart action potential. Certainmutations in either KCNQ1 or KCNE1 cause a form of the autosomaldominant long QT syndrome (LQTS), a disease characterised byrepolarization anomalies of cardiac action potentials resulting inarrhythmias and sudden death. Interestingly, other mutations in eithergene lead to the recessive Jervell and Lange-Nielsen (JLN) syndrome thatcombines LQTS with congenital deafness. In order to cause deafness,KCNQ1/minK currents must be reduced below levels that are alreadysufficiently low to cause cardiac arrhythmia.

[0005] Mutated and non-mutated KCNQ2 and KCNQ3 potassium channels havebeen disclosed in WO 99/07832, WO 99/21875 and WO 99/31232.

SUMMARY OF THE INVENTION

[0006] We have now cloned and characterised KCNQ5, a novel member of theKCNQ family of potassium channel proteins. KCNQ5 forms heteromericchannels with other KCNQ channel subunits, in particular KCNQ3 andKCNQ4.

[0007] The present invention has important implications for thecharacterisation and exploitation of this interesting branch of thepotassium channel super family.

[0008] Accordingly, in its first aspect, the invention provides anisolated polynucleotide having a nucleic acid sequence which is capableof hybridising under at least medium stringency conditions with thepolynucleotide sequence presented as SEQ ID NO: 1, its complementarystrand, or a sub-sequence thereof.

[0009] In another aspect the invention provides a recombinantly producedpolypeptide encoded by the polynucleotide of the invention.

[0010] In a third aspect the invention provides a cell geneticallymanipulated by the incorporation of a heterologous polynucleotide of theinvention.

[0011] In a fourth aspect the invention provides a method of screening achemical compound for inhibiting or activating or otherwise modulatingthe activity on a potassium channel comprising at least one KCNQ5channel subunit, which method comprises the steps of subjecting a KCNQ5channel subunit containing cell to the action of the chemical compound;and monitoring the membrane potential, the current, the potassium flux,or the secondary calcium influx of the KCNQ5 channel subunit containingcell.

[0012] In a fifth aspect the invention relates to the use of apolynucleotide sequence of the invention for the screening of geneticmaterials from humans suffering from neurological diseases for mutationsin the KCNQ5 gene.

[0013] In a sixth aspect the invention relates to the chemical compoundidentified by the method of the invention, in particular to the use ofsuch compounds for diagnosis, treatment or alleviation of a diseaserelated to diseases or adverse conditions of the CNS, includingaffective disorders, Alzheimer's disease, anxiety, ataxia, CNS damagecaused by trauma, stroke or neurodegenerative illness, cognitivedeficits, compulsive behaviour, dementia, depression, Huntington'sdisease, mania, memory impairment, memory disorders, memory dysfunction,motion disorders, motor disorders, neurodegenerative diseases,Parkinson's disease and Parkinson-like motor disorders, phobias, Pick'sdisease, psychosis, schizophrenia, spinal cord damage, stroke, tremor,seizures, convulsions and epilepsy.

[0014] In a seventh aspect the invention provides a transgenic animalcomprising a knock-out mutation of the endogenous KCNQ5 gene, areplacement by or an additional expression of a mutated KCNQ5 gene, orgenetically manipulated in order to over-express the KCNQ5 gene or toover-express mutated KCNQ5 gene.

[0015] In an eighth aspect the invention relates to the use of thetransgenic animal of the invention for the in vivo screening oftherapeutic compounds.

[0016] Other objects of the invention will be apparent to the personskilled in the art from the following detailed description and examples.

DETAILED DISCLOSURE OF THE INVENTION

[0017] The present invention provides novel potassium channels and genesencoding these channels. The invention also provides cells transformedwith these genes, transgenic animals comprising genetic mutations, andthe use of the transformed cells and the transgenic animals for the invitro and in vivo screening of drugs affecting KCNQ5 containingpotassium channels.

[0018] Polynucleotides

[0019] In its first aspect, the invention relates to novel nuceic acidmolecules encoding a polypeptide comprising all or a portion of a KCNQ5protein.

[0020] In a preferred embodiment, the polynucleotides of the inventionare such which have a nucleic acid sequence capable of hybridising underat least medium stringency conditions with the polynucleotide sequencepresented as SEQ ID NO: 1, its complementary strand, or a sub-sequencethereof.

[0021] The polynucleotides of the invention include DNA, cDNA and RNAsequences, as well as anti-sense sequences, and include naturallyoccurring, synthetic, and intentionally manipulated polynucleotides. Thepolynucleotides of the invention also include sequences that aredegenerate as a result of the genetic code. As defined herein, the term“polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length, preferably at least 15 bases in length. By “isolatedpolynucleotide” is meant a polynucleotide that is not immediatelycontiguous with both of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Theterm therefore includes recombinant DNA which is incorporated into anexpression vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule, e.g. a cDNA, independent from other sequences.

[0022] The polynucleotides of the invention also include allelicvariants and “mutated polynucleotides” having a nucleotide sequence thatdiffers from the sequence presented as SEQ ID NO: 1 at one or morenucleotide positions. The mutated polynucleotide may in particular be apolynucleotide of the invention having a nucleotide sequence as in SEQID NO: 1, which sequence, however, differs from SEQ ID NO: 1 so as toeffect the expression of a variant polypeptide. The mutatedpolynucleotide may be a polynucleotide of the invention having anucleotide sequence encoding a potassium channel having an amino acidsequence that has been changed at one or more positions. The mutatedpolynucleotide may in particular be a polynucleotide of the inventionhaving a nucleotide sequence encoding a potassium channel having anamino acid sequence that has been changed at one or more positionslocated in the conserved regions, as defined by Table 1, below.

[0023] Hybridisation Protocol

[0024] The polynucleotides of the invention are such which have anucleic acid sequence capable of hybridising with the polynucleotidesequence presented as SEQ ID NO: 1, its complementary strand, or asub-sequence thereof, under at least medium, medium/high, or highstringency conditions, as described in more detail below.

[0025] In a preferred embodiment the polynucleotide is a fragment of atleast 15 bases in length which is sufficient to permit the fragment tohybridise to DNA that encodes a polypeptide of the invention, preferablythe polypeptide having the amino acid sequence presented as SEQ ID NO: 2under at least medium, medium/high, or high stringency conditions, asdescribed in more detail below.

[0026] Suitable experimental conditions for determining hybridisationbetween a nucleotide probe and a homologous DNA or RNA sequence,involves pre-soaking of the filter containing the DNA fragments or RNAto hybridise in 5×SSC [Sodium chloride/Sodium citrate; cf. Sambrook etal.; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab.,Cold Spring Harbor, N.Y. 1989] for 10 minutes, and pre-hybridisation ofthe filter in a solution of 5×SSC, 5× Denhardt's solution [cf. Sambrooket al.; Op cit.], 0.5% SDS and 100 μg/ml of denatured sonicated salmonsperm DNA [cf. Sambrook et al.; Op cit.], followed by hybridisation inthe same solution containing a concentration of 10 ng/ml of arandom-primed [Feinberg A P & Vogelstein B; Anal. Biochem. 1983 1326-13], ³²P-dCTP-labeled (specific activity >1×10⁹ cpm/μg) probe for 12hours at approximately 45° C.

[0027] The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDSat a temperature of at least at least 60° C. (medium stringencyconditions), preferably of at least 65° C. (medium/high stringencyconditions), more preferred of at least 70° C. (high stringencyconditions), and even more preferred of at least 75° C. (very highstringency conditions).

[0028] Molecules to which the oligonucleotide probe hybridises underthese conditions may be detected using a x-ray film.

[0029] DNA Sequence Homology

[0030] In a preferred embodiment, the polynucleotides of the inventionshow a homology of at least 65%, preferably at least 70%, more preferredat least 80%, even more preferred at least 90%, most preferred at least95%, with the polynucleotide sequence presented as SEQ ID NO: 1.

[0031] As defined herein, the DNA sequence homology may be determined asthe degree of identity between two DNA sequences indicating a derivationof the first sequence from the second. The homology may suitably bedetermined by means of computer programs known in the art such as GAPprovided in the GCG program package [Needleman S B and Wunsch C D,Journal of Molecular Biology 1970 48 443-453] using default parameterssuggested herein.

[0032] Cloned Polynucleotides

[0033] The isolated polynucleotide of the invention may in particular bea cloned polynucleotide.

[0034] As defined herein, the term “cloned polynucleotide”, refers to apolynucleotide or DNA sequence cloned in accordance with standardcloning procedures currently used in genetic engineering to relocate asegment of DNA, which may in particular be cDNA, i.e. enzymaticallyderived from RNA, from its natural location to a different site where itwill be reproduced.

[0035] Cloning may be accomplished by excision and isolation of thedesired DNA segment, insertion of the piece of DNA into the vectormolecule and incorporation of the recombinant vector into a cell wheremultiple copies or clones of the DNA segment will be replicated, byreverse transcription of mRNA (reverse transcriptase technology), and byuse of sequence-specific oligonucleotides and DNA polymerase in apolymerase chain reaction (PCR technology).

[0036] The cloned polynucleotide of the invention may alternatively betermed “DNA construct” or “isolated DNA sequence”, and may in particularbe a complementary DNA (cDNA).

[0037] It is well established that potassium channels may be formed as.heteromeric channels, composed of different subunits. Also it has beenestablished that the potassium channel of the invention may formheteromers with other KCNQ's, in particular KCNQ3 and KCNQ4, whenco-expressed with these subunits. In addition, potassium channels canassociate with non-homologous subunits (β-subunits), e.g. the KCNE1(also known as minK or IsK), the KCNE2 (also known as the minK-relatedpeptide (MiRP1), the KCNE3 (also known as MiRP2), the KCNE4 (also knownas MiRP3), and/or the KCNE5 (also known as KCNE1L) subunit, thatco-assemble and functionally modulate these channels or lead to aspecific localisation within the cell.

[0038] Therefore, in a preferred embodiment, the polynucleotide of theinvention is cloned and either expressed by itself or co-expressed withpolynucleotides encoding other subunits, in particular a polynucleotideencoding a KCNQ3 channel subunit or a polynucleotide encoding a KCNQ4channel subunit.

[0039] In another aspect of the invention, isolated and purified KCNQ5antisence oligonucleotides can be made and a method utilised fordiminishing the level of expression of KCNQ5 by a cell comprisingadministering one or more KCNQ5 antisense oligonucleotides. By KCNQ5antisense oligonucleotides reference is made to oligonucleotides thathave a nucleotide sequence that interacts through base pairing with aspecific complementary nucleic acid sequence involved in the expressionof KCNQ5 such that the expression of KCNQ5 is reduced. Preferably, thenucleic acid sequence involved in the expression of KCNQ5 is a genomicDNA molecule or mRNA molecule that encodes KCNQ5. This genomic DNAmolecule can comprise regulatory regions of the KCNQ5 gene, the pre- orpro-portions of the is KCNQ5 gene, or the coding sequence for matureKCNQ5 protein.

[0040] The term “complementary to a nucleotide sequence” in the contextof KCNQ5 antisense oligonucleotides and methods therefor meanssufficiently complementary to such a sequence as to allow hybridizationto that sequence in a cell, i.e. under physiological conditions. TheKCNQ5 antisense oligonucleotides preferably comprise a sequencecomprising of from about 8 to about 100 nucleotides, more preferably offrom about 15 to about 30 nucleotides.

[0041] The KCNQ5 antisense oligonucleotides can also include derivativeswhich comprise a variety of modifications that confer resistance tonucleolytic degradation such as e.g. modified internucleoside linkagesmodified nucleic acid bases and/or sugars and the like. Examples of suchderivatives include backbone modifications such as phosphotriester,phosphorothioate, methylphosphate, phosphoramidate, phosphorodithioateand formacetal as well as morpholino, peptide nucleic acid analogues anddithioate repeating units.

[0042] The usefulness of antisense molecules have been described by e.g.Toulme & Helene, Gene 1988 72 51-58; Inouye, Gene 1988 72 25-34; Uhlmann& Peyman, Chemical Reviews 1990 90 543-584; Robertson, NatureBiotechnology 1997 15 209; and Gibbons & Dzau, Science 1996 272 689-693,which publications are hereby incorporated by reference.

[0043] Biological Sources

[0044] The isolated polynucleotide of the invention may be obtained fromany suitable human or animal source. In a preferred embodiment, whichthe polynucleotide of the invention is cloned from, or produced on thebasis of a cDNA library, e.g. of the retina, skeletal muscle, or brain,in particular the cerebral cortex, occipital pole, frontal and temporallobes, putamen and the hippocampus, and in the piriform cortex, theentorhinal cortex, the pontine medulla and the facial nucleus, and inthe cerebellum. In a more preferred embodiment, which the polynucleotideof the invention is cloned from, or produced on the basis of a cDNAlibrary of the thalamus. Commercial cDNA libraries are available frome.g. Stratagene and Clontech.

[0045] The KCNQ5 gene of the invention has been localised to the longarm of chromosome 6 (6q14).

[0046] The isolated polynucleotide of the invention may be obtainedmethods known in the art, e.g. those described in the working examplesbelow.

[0047] Preferred Polynucleotides

[0048] In a preferred embodiment, polynucleotide of the invention hasthe polynucleotide sequence presented as SEQ ID NO: 1.

[0049] In another preferred embodiment the polynucleotide of theinvention is a sequence giving rise to KCNQ5 channels subunitscomprising one or more substitutions.

[0050] In another preferred embodiment the polynucleotide of theinvention is a sequence giving rise to KCNQ5 channels subunitscomprising one or more substitutions in the conserved regions, asdefined in more details below.

[0051] It has been demonstrated that KCNQ channels often showalternative splicing and therefore may occur as isoforms originatingfrom the same gene. Such isoforms as well as the different cDNAsequences from which they occurred are also contemplated within thescope of the present invention.

[0052] Finally the genes encoding KCNQ channel subunits in other specieshave been found to differ slightly from the human genes. However, genesof other species, e.g. mouse, rat, monkey, rabbit, etc., are alsocontemplated within the scope of the present invention.

[0053] Recombinantly Produced Polypeptides

[0054] In another aspect the invention relates to novel KCNQ5 proteins.More. specifically, the invention relates to substantially purefunctional polypeptides that have the electrophysiological andpharmacological properties of a KCNQ5 channel, or KCNQ5 channelsubunits. The novel polypeptides of the invention may be obtained by thepolynucleotides of the invention using standard recombinant DNAtechnology.

[0055] In a preferred embodiment, a polypeptide of the invention is theKCNQ5 potassium channel having the amino acid sequence presented as SEQID NO: 2, and biologically active fragments hereof.

[0056] Modifications of this primary amino acid sequence may result inproteins which have substantially equivalent activity as compared to theunmodified counterpart polypeptide, and thus may be consideredfunctional analogous of the parent proteins. Such modifications may bedeliberate, e.g. as by site-directed mutagenesis, or they may occurspontaneous, and include splice variants, isoforms, homologues fromother species, and polymorphisms. Such functional analogous are alsocontemplated according to the invention.

[0057] Moreover, modifications of this primary amino acid sequence mayresult in proteins which do not retain the biological activity of theparent protein, including dominant negative forms, etc. A dominantnegative protein may interfere with the wild-type protein by binding to,or otherwise sequestering regulating agents, such as upstream ordownstream components, that normally interact functionally with thepolypeptide. Such dominant negative forms are also contemplatedaccording to the invention.

[0058] In the context of this invention, the term “variant polypeptide”means a polypeptide (or protein) having an amino acid sequence thatdiffers from the sequence presented as SEQ ID NO: 2 at one or more aminoacid positions. Such variant polypeptides include the modifiedpolypeptides described above, as well as conservative substitutions,splice variants, isoforms, homologues from other species, andpolymorphisms.

[0059] As defined herein, the term “conservative substitutions” denotesthe replacement of an amino acid residue by another, biologicallysimilar residue. Examples of conservative substitutions include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine or methionine for another, or the substitution of one polarresidue for another, such as the substitution of arginine for lysine,glutamic for aspartic acid, or glutamine for asparagine, and the like.The term conservative substitution also include the use of a substitutedamino acid residue in place of an un-substituted parent amino acidresidue provided that antibodies raised to the substituted polypeptidealso immune-react with the un-substituted polypeptide.

[0060] KCNQ1 Numbering System

[0061] In the context of this invention, amino acid residues (as well asnucleic acid bases) are specified using the established one-lettersymbol.

[0062] By aligning the amino acid sequences of a polypeptide of thepresent invention to those of the known polypeptides, a specific aminoacid numbering system may be employed, by which system it is possible tounambiguously allot an amino acid position number to any amino acidresidue in any KNCQ channel protein, which amino acid sequence is known.

[0063] Such an alignment is presented in Table 1, below. Using theClustalX computer alignment program [Thompson J D, Gibson T J, PlewniakF, Jeanmougin F, & Higgins D G: The ClustalX windows interface: flexiblestrategies for multiple sequence alignment aided by quality analysistools; Nucleic Acids Res. 1997 25 (24) 4876-82], and the defaultparameters suggested herein, the amino acid sequence of a polypeptide ofthe present invention (hKCNQ5) and the amino acid sequences of the knownpolypeptides hKCNQ2-4 are aligned with, and relative to, the amino acidsequences of the known polypeptide hKCNQ1 (also known as KvLQT1). In thecontext of this invention this numbering system is designated the KCNQ1Numbering System.

[0064] In describing the various enzyme variants produced orcontemplated according to the invention, the following nomenclatureshave been adapted for ease of reference:

Original amino acid/Position/Substituted amino acid

[0065] According to this nomenclature the substitution of serine forglycine at position 329 of Table 1 is designated as “G329S”. TABLE 1CLUSTAL X Multiple Sequence Alignment KCNQ1 Numbering hKCNQ1 MAAASSPPRAE-----RKRW GWGRLPGARR GSAGLAKKCP FSLELAEGG- ---P--AGGA 60 hKCNQ2MVQKSR---- ---------- NGGVYPGPSG EKKLKVG--- -FVGLDPG-- -------APD hKCNQ3MGLKARRAAG AAGGGGDGGG GGGGAANPAG GDAAAAGDEE RKVGLAPGDV EQVTLALGAG hKCNQ4MAEAPPRR-- ---------L GLGPPPGDAP RAELVALT-- -AVQSEQGE- -------AGG hKCNQ5MKDVES---- ---------- GRGRVLLNSA AARQDGLLLL GTRAATLGG- -------GGG*                       *                          * hKCNQ1 LYAPIAPGAPGPAPPASPAA PAAPPVASDL GPRPPVSLDP RVSIYSTRRP VLARTHVQGR 120 hKCNQ2STRDGALLIA G-----SEAP KRGSILSKPR AGGAGAGKPP KRN-AFYRK- ------LQNF hKCNQ3ADKDGTLLLE GGG---RDEG QRRTPQGIGL LAKTPLSRPV KRNNAKYRR- ------IQTL hKCNQ4GGSPRRLGLL G-----SPLP PGAPLPGPGS GSGSACGQRS SAAHKRYRR- ------LQNW hKCNQ5GLRESRRGKQ G--------- ARMSLLGKPL SYTS--SQSC RRN-VKYRR- ------VQNY           *                                       *          * hKCNQ1VYNFLERPTG WKCFVYHFAV FLIVLVCLIF SVLSTIEQYA ALATGTLFWM EIVLVVFFGT 180hKCNQ2 LYNVLERPRG W-AFIYHAYV FLLVFSCLVL SVFSTIKEYE KSSEGALYIL EIVTIVVFGVhKCNQ3 IYDALERPRG W-ALLYHALV FLIVLGCLIL AVLTTFKEYE TVSGDWLLLL ETFAIFIFGAhKCNQ4 VYNVLERPRG W-AFVYHVFI FLLVFSCLVL SVLSTIQEHQ ELANECLLIL EFVMIVVFGLhKCNQ5 LYNVLERPRG W-AFIYHAFV FLLVFGCLIL SVFSTIPEHT KLASSCLLIL EFVMIVVFGL *  **** * *    **    ** *  **    *  *            *    *      **                         Site 1                     Site 2        hKCNQ1EYVVRLWSAG CRSKYVGLWG RLRFARKPIS IIDLIVVVAS MVVLCVGSKG QVFATSAIRG 240hKCNQ2 EYFVRIWAAG CCCRYRGWRG RLKFARKPFC VIDIMVLIAS IAVLAAGSQG NVFATSALRShKCNQ3 EFALRIWAAG CCCRYKGWRG RLKFARKPLC MLDIFVLIAS VPVVAVGNQG NVLATS-LRShKCNQ4 EYIVRVWSAG CCCRYRGWQG RFRFARKPFC VIDFIVFVAS VAVIAAGTQG NIFATSALRShKCNQ5 EFIIRIWSAG CCCRYRGWQG RLRFARKPFC VIDTIVLIAS IAVVSAKTQG NIFATSALRS*   * * ** *   * *  * *  *****     *  *  **  *       *    ***  *                                      Site 3                 Site hKCNQ1IRFLQILRML HVDRQGGTWR LLGSVVFIHR QELITTLYIG FLGLIFSSYF VYLAEKDAVN 300hKCNQ2 LRFLQILRMI RMDRRGGTWK LLGSVVYAHS KELVTAWYIG FLCLILASFL VYLAEK----hKCNQ3 LRFLQILRML RMDRRGGTWK LLGSAICAHS KELITAWYIG FLTLILSSFL VYLVEKDVPEhKCNQ4 MRFLQILRMV RMDRRGGTWK LLGSVVYAHS KELITAWYIG FLVLIFASFL VYLAEKD---hKCNQ5 LRFLQILRMV RMDRRGGTWK LLGSVVYAHS KELITAWYIG FLVLIFSSFL VYLVEKD---  *******    ** ****  ****    *   ** *  *** ** **  *   *** **4                                          Site 5           hKCNQ1-----ESGRV EFGSYADALW WGVVTVTTIG YGDKVPQTWV GKTIASCFSV FAISFFALPA 360hKCNQ2 ----GE--ND HFDTYADALW WGLITLTTIG YGDKYPQTWN GRLLAATFTL IGVSFFALPAhKCNQ3 VDAQGEEMKE EFETYADALW WGLITLATIG YGDKTPKTWE GRLIAATFSL IGVSFFALPAhKCNQ4 -------ANS DFSSYADSLW WGTITLTTIG YGDKTPHTWL GRVLAAGFAL LGISFFALPAhKCNQ5 -------ANK EFSTYADALW WGTITLTTIG YGDKTPLTWL GRLLSAGFAL LGISFFALPA            *  *** ** **  *  *** **** * **  *      *     ********                        P-loop                           Site 6   hKCNQ1GILGSGFALK VQQKQRQKHF NRQIPAAASL IQTAWRCYAA E---NPDSST WKIYIRKAP- 420hKCNQ2 GILGSGFALK VQEQHRQKHF EKRRNPAAGL IQSAWRFYAT NLSRTDLHST WQYYERTVT-hKCNQ3 GILGSGLALK VQEQHRQKHF EKRRKPAAEL IQAAWRYYAT NPNRIDLVAT WRFYESVVS-hKCNQ4 GILGSGFALK VQEQHRQKHF EKRRMPAANL IQAAWRLYST DMSRAYLTAT WYYYDSILPShKCNQ5 GILGSGFALK VQEQHRQKHF EKRRNPAANL IQCVWRSYAA D-EKSVSIAT WKPHLKALHT****** *** **   ****        ** * **  ** *            * *        hKCNQ1-------RSH TLLS------ PSPKPK---- ---------- ---------- -----KSVVV 480hKCNQ2 -------VPM YRLIPP--LN QLELLRNLKS KSGLAFRK-- -------DPP PEPSPSQKVShKCNQ3 -------FPF FRKE------ QLEAAS---- ---------- ---------- ---S--QKLGhKCNQ4 FRELALLFEH VQRARNGGLR PLEVRRAPVP DGAPSRYPPV ATCHRPGSTS FCPGESSRMGhKCNQ5 -------CSP TKKE------ QGEASS---- ---------- ---------- -----SQKLS                           Alternatively Spliced hKCNQ1 KKKKPKLDKDNGVTPGEKML TVPH-ITCDP PEERRLDHFS VDGYDSSVRK SPTLLEVS-M 540 hKCNQ2LKDRV-FSSP RGVAAKGKGS PQAQTVRRSP SADQSLED-- ---SPSKVPK SWSFGDRSRA hKCNQ3LLDRVRLSNP RGSNTKGK-- ------LFTP LNVDAIEE-- ---SPSKEPK PVGLNNKERF hKCNQ4IKDRIRMGSS QRRTGPSKQQ LAPPTMPTSP SSEQVGEAT- ---SPTKVQK SWSFNDRTRF hKCNQ5FKERVRMASP RGQSIKSRQA SVGD--RRSP STDITAEG-- ---SPTKVQK SWSFNDRTRP                               *                     * hKCNQ1 PHFMRTNS------FAEDLD LEGETLLTPI TH-----ISQ LREHHRATIK VIRRMQYFVA 600 hKCNQ2RQAFRIKGAA S-RQNSEEAS LPGEDIVDDK SCPCEFVTED LTPGLKVSIR AVCVMRFLVS hKCNQ3RTAFRMKAYA F-WQSSEDAG T-GDPMAEDR GYGNDFPIED MIPTLKAAIR AVRILQFRLY hKCNQ4RASLRLKP-- --RTSAEDAP S--EEVAEEK SYQCELTVDD IMPAVKTVIR SIRILKFLVA hKCNQ5RPSLRLKSSQ PKPVIDADTA LGTDDVYDEK GCQCDVSVED LTPPLKTVIR AIRIMKFHVA     *                                                *                                                                 hKCNQ1KKKFQQARKP YDVRDVIEQY SQGHLNLMVR IKELQRRLDQ SIGK-PSLFI SVS--EKS-- 660hKCNQ2 KRKFKESLRP YDVMDVIEQY SAGHLDMLSR IKSLQSRVDQ IVGRGPAITD KDR--TKG--hKCNQ3 KKKFKETLRP YDVKDVIEQY SAGHLDMLSR IKYLQTRIDM IFTPGPPSTP KHKKSQKGSAhKCNQ4 KRKFKETLRP YDVKDVIEQY SAGHLDMLGR IKSLQTRVDQ IVGRGFGDRK AREKGDKG--hKCNQ5 KRKFKETLRP YDVKDVIEQY SAGHLDMLCR IKSLQTRVDQ ILGKGQITSD KKSREKIT--* **     * *** ****** * ***    * ** ** *  *          A-domain                                 hKCNQ1 -------------------K DRG--SNTIG ARLNRVEDKV TQLDQRLALI TDMLHQLLSL 720 hKCNQ2---------- -------PAE AELPEDPSMM GRLGKVEKQV LSMEKKLDFL VNIYMQRMGI hKCNQ3FTFPSQQSPR NEPYVARPST SEI-EDQSMM GKFVKVERQV QDMGKKLDFL VDMHMQHMER hKCNQ4---------- -------PSD AEVVDEISMM GRVVKVEKQV QSIEHKLDLL LGFYSRCLRS hKCNQ5---------- --------AE HETTDDLSML GRVVKVEKQV QSIESKLDCL LDIYQQVLRK                                      **  *       * hKCNQ1 HGGSTPG---SGGPPREGG- -AHITQPCGS G--GSVDPEL FLPSNTLPTY EQLTVP-RRG 780 hKCNQ2PPTETE---- AYFGAKEPEP APPYNSPEDS REHVDRHGCI VKIVRSSSST GQKNF----S hKCNQ3LQVQVT---- EYYPTKGTSS PAEAEKKEDN R-YSDLKTII CNYSETGPPE PPYSFH-QVT hKCNQ4GTSASLGA-- VQVPLFDPDI TSDYHSPVDH E-DISVSAQT LSISRSVSTN MD-------- hKCNQ5GSASALALAS FQIPPFECEQ TSDYQSPVDS KDLSGSAQNS GCLSRSTSAN ISRGLQFILT hKCNQ1PDEGS----- ---------- ---------- ---------- ---------- ---------- 840hKCNQ2 APPAAPPVQC PPSTSWQPQS HPRQGH---- ---------- ---GTSFVGD HGSLVRIPPPhKCNQ3 IDKVSPYGFF AHDPVNLPRG GPSSGKV--- ---------- ---QATPPSS ATTYVERPTVhKCNQ4 ---------- ---------- ---------- ---------- ---------- ----------hKCNQ5 PNEFSAQTFY ALSPTMHSQA TQVPISQSDG SAVAATNTIA NQINTAPKPA APTTLQIPPPhKCNQ1 ---------- ---------- ---------- ---------- ---------- ----------900 hKCNQ2 PAHERSLSAY GGGN-RASME FLRQEDTPGC R-PPEGTLRD SDTSISIPSVDHEELERSFS hKCNQ3 LPILTLLDSR VSCH-SQADL QGPYSDRISP R-QRRSITRD SDTPLSLMSVNHEELERSPS hKCNQ4 ---------- ---------- ---------- ---------- -------------------- hKCNQ5 LPAIKHLPRP ETLHPNPAGL QESISDVTTC LVASKENVQV AQSNLTKDRSMRKSFDMGGE hKCNQ1 ---------- ---------- ---------- ---------- -------------------- 960 hKCNQ2 GFSISQSKEN LDALNSCYAA VAPCAKVRPY IAEGESDTD-----SDLCTP CGPPPRSATG hKCNQ3 GFSISQDRDD YVFGPNGGSS WM---REKRY LAEGETDTD-----TDPFTP SGSMPLSSTG hKCNQ4 ---------- ---------- ---------- -------------------- ---------- hKCNQ5 TLLSVCPMVP KDLGKSLSVQ NLIRSTEELN IQLSGSESSGSRGSQDFYPK WRESKLFITD hKCNQ1 ---------- ------ 976 hKCNQ2 EGPFGDVGWAGPRK-- hKCNQ3 DG-ISDSVWT PSNKPI hKCNQ4 ---------- ------ hKCNQ5EEVGPEETET DTFARI+TZ,1 47

[0066] hKCNQ1: Human KCNQ1 [Wang, Q et al.; Nature Genet. 1996 12 17-23]

[0067] hKCNQ2: Human KCNQ2 [Biervert et al.; Science 1998 279 403-406]

[0068] hKCNQ3: Human KCNQ3 [Schroederetal.; Nature 1998 396 687-690]

[0069] hKCNQ4: Human KCNQ4 [Kubisch et al.; Cell 1999 96 (3) 437-46]

[0070] hKCNQ5: Human KCNQ5; A protein of the invention

[0071] No amino acid in this position.

[0072] Indicates positions which holds a single, fully conserved residue(Conserved regions).

[0073] Preferred variants are the splice variants at positions 432-476(KCNQ1 Numbering) holding the following amino acid residues: 1)KKE------ QGEASS---- ------NKFC SNKQKLFRMY TSRKQS; 2) KKE------QGEASS---- ---------- ---------- ------; 3) --------- -------------------- ---------- ------; or 4) --------- ---------- ------NKFCSNKQKLFRMY TSRKQS. Another preferred variants is G329S (KCNQ1numbering), or KCNQ5  G278S (“KCNQ5 numbering”).

Biological Activity

[0074] Ion channels are excellent targets for drugs. The polynucleotideof the invention encodes a potassium channel, which has been termedKNCQ5.

[0075] KCNQ5, or heteromeric channels containing the KCNQ5 subunit, maybe a particularly interesting target for the treatment of diseases oradverse conditions of the CNS, including affective disorders,Alzheimer's disease, anxiety, ataxia, CNS damage caused by trauma,stroke or neurodegenerative illness, cognitive deficits, compulsivebehaviour, dementia, depression, Huntington's disease, learningdeficiencies, mania, memory impairment, memory disorders, memorydysfunction, motion disorders, motor disorders, motor neuron doseases,myokymia, neurodegenerative diseases, Parkinson's disease andParkinson-like motor disorders, phobias, Pick's disease, psychosis,schizophrenia, seizures, incl. epileptic seizures, spinal cord damage,stroke, tremor, seizures, convulsions and epilepsy.

[0076] The novel polynucleotides of the invention may in itself be usedas a therapeutic or diagnostic agent. For gene therapy, the personskilled in the art may use sence or antisense nucleic acid molecules astherapeutic agents for KCNQ-related indications.

[0077] Heteromers Formed by KCNQ Subunits

[0078] The KCNQ channels described so far function physiologically asheteromers. KCNQ1 associates with KCNE1 (also known as mink or IsK);KCNQ2 and KCNQ3 form heteromeric channels that underlie the M-current,an important determinant of neuronal excitability that is regulated byseveral neurotransmitters, and KCNQ4 is supposed to combine with KCNQ3to mediate the I_(M)-like current in the outer hair cells.

[0079] Like other KCNQ channel subunits, KCNQ5 may interact with othersubunits, e.g. KCNE1 or other KCNQ channel subunits, and in particularwith KCNQ3, and with KCNQ4. Currents from homomeric KCNQ3 are very smalland often cannot be distinguished from Xenopus oocyte backgroundcurrents. Co-expression of KCNQ3 with KCNQ5 markedly increased currentamplitudes. Co-expression of KCNQ4 with KCNQ5 markedly decreased currentamplitudes.

[0080] Antibodies

[0081] The polypeptides of the invention can be used to produceantibodies which are immunoreactive or bind to epitopes of thesepolypeptides. Polyclonal antibodies which consist essentially of pooledmonoclonal antibodies with different specificities, as well as distinctmonoclonal antibody preparations may be provided. Polyclonal antibodieswhich are made up of pooled monoclonal antibodies with differentspecificities, as well as distinct monoclonal antibody preparations maybe provided.

[0082] The preparation of polyclonal and monoclonal antibodies is wellknown in the art. Polyclonal antibodies may in particular be obtained asdescribed by e.g. Green et al.: “Production of Polyclonal Antisera” inImmunochemical Protocols (Manson, Ed.); Humana Press, 1992, Pages 1-5;Coligan et al.: “Production of Polyclonal Antisera in rabbits, rats,Mice and Hamsters” in Current Protocols in Immunology, 1992, Section2.4.1; and Ed Harlow and David Lane (Eds.) in “Antibodies; A laboratorymanual”, Cold Spring Harbor Lab Press 1988; which protocols are herebyincorporated by reference.

[0083] Monoclonal antibodies may in particular be obtained as describedby e.g. Kohler & Milstein, Nature 1975 256 495; Coligan et al. inCurrent Protocols in Immunology, 1992, Sections 2.5.1-2.6.7; Hadlow etal. in Antibodies: A Laboratory Manual; Cold Spring Harbor Pub., 1988,Page 726; which protocols are hereby incorporated by reference.

[0084] Briefly, monoclonal antibodies may be obtained by injecting e.g.mice with a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce the antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

[0085] Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques, including affinitychromatography with protein A Sepharose, size-exclusion chromatography,and ion-exchange chromatography, see. e.g. Coligan et al. in CurrentProtocols in Immunology, 1992, Sections 2.7.1-2.7.12, and Sections2.9.1-2.9.3; and Bames et al.: “Purification of Immunoglobulin G (IgG)”in Methods in Molecular Biology; Humana Press, 1992, Vol. 10, Pages79-104.

[0086] The polyclonal or monoclonal antibodies may optionally be furtherpurified, e.g. by binding to and elution from a matrix to which thepolypeptide, to which the antibodies were raised, is bound.

[0087] Antibodies which bind to the polypeptide of the invention can beprepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunising antigen. The polypeptide used toimmunise an animal may be obtained by recombinant DNA techniques or bychemical synthesis, and may optionally be conjugated to a carrierprotein. Commonly used carrier proteins which are chemically coupled tothe peptide include keyhole limpet hemocyanin (KLH), thyroglobulin,bovine serum albumin (BSA), and tetanus toxoid. The coupled peptide maythen be used to immunise the animal, which may in particular be a mouse,a rat, a hamster or a rabbit.

[0088] Genetically Manipulated Cells

[0089] In a third aspect the invention provides a cell geneticallymanipulated by the incorporation of the heterologous polynucleotide ofthe invention. The cell of the invention may in particular begenetically manipulated to transiently or stably express, over-expressor co-express a KCNQ5 channel subunit as defined above. Methods oftransient and stable transfer are known in the art.

[0090] The polynucleotide of the invention may be inserted into anexpression vector, e.g. a plasmid, virus or other expression vehicle,and operatively linked to expression control sequences by ligation in away that expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. Suitable expressioncontrol sequences include promoters, enhancers, transcriptionterminators, start codons, splicing signals for introns, and stopcodons, all maintained in the correct reading frame of thepolynucleotide of the invention so as to permit proper translation ofmRNA. Expression control sequences may also include additionalcomponents such as leader sequences and fusion partner sequences.

[0091] The promoter may in particular be a constitutive or an induciblepromoter. When cloning in bacterial systems, inducible promoters such aspL of bacteriophage γ, plac, ptrp, ptac (ptrp-lac hybrid promoter), maybe used. When cloning in mammalian systems, promoters derived from thegenome of mammalian cells, e.g. the TK promoter or the metallothioneinpromoter, or from mammalian viruses, e.g. the retrovirus long terminalrepeat, the adenovirus late promoter or the vaccinia virus 7.5Kpromoter, may be used. Promoters obtained by recombinant DNA orsynthetic techniques may also be used to provide for transcription ofthe polynucleotide of the invention.

[0092] Suitable expression vectors typically comprise an origin ofexpression, a promoter as well as specific genes which allow forphenotypic selection of the transformed cells, and include vectors likethe T7-based expression vector for expression in bacteria [Rosenberg etal; Gene 1987 56 125], the pMSXND expression vector for expression inmammalian cells [Lee and Nathans, J. Biol. Chem. 1988 263 3521],baculovirus derived vectors for expression in insect cells, and theoocyte expression vector PTLN [Lorenz C, Pusch M & Jentsch T J:Heteromultimeric CLC chloride channels with novel properties; Proc.Natl. Acad. Sci. USA 1996 93 13362-13366].

[0093] In a preferred embodiment, the cell of the invention is aneukaryotic cell, in particular a mammalian cell, an oocyte, or a yeastcell. In a more preferred embodiment, the cell of the invention is ahuman embryonic kidney (HEK) cell, a HEK 293 cell, a BHK21 cell, aChinese hamster ovary (CHO) cell, a Xenopus laevis oocyte (XLO) cell, aCOS cell, or any other cell line able to express KCNQ potassiumchannels.

[0094] When the cell of the invention is an eukaryotic cell,incorporation of the heterologous polynucleotide of the invention may bein particular be carried out by infection (employing a virus vector), bytransfection (employing a plasmid vector), or by calcium phosphateprecipitation, microinjection, electroporation, lipofection, or otherphysical-chemical methods known in the art.

[0095] In a further preferred embodiment, the cell of the invention isgenetically manipulated to co-express KCNQ5 and KCNQ1 channel subunits;KCNQ5 and KCNQ2 channel subunits; KCNQ5 and KCNQ3 channel subunits;KCNQ5 and KCNQ4 channel subunits; KCNQ5 and KCNQ1 and KCNQ2 channelsubunits; KCNQ5 and KCNQ1 and KCNQ3 channel subunits; KCNQ5 and KCNQ2and KCNQ3 channel subunits; KCNQ5 and KCNQ1 and KCNQ4 channel subunits;KCNQ5 and KCNQ2 and KCNQ4 channel subunits; KCNQ5 and KCNQ3 and KCNQ4channel subunits; KCNQ5 and KCNQ1 and KCNQ2 and KCNQ3 channel subunits;KCNQ5 and KCNQ1 and KCNQ2 and KCNQ4 channel subunits; KCNQ5 and KCNQ1and KCNQ3 and KCNQ4 channel subunits, or KCNQ5 and KCNQ2 and KCNQ3 andKCNQ4 channel subunits.

[0096] In another preferred embodiment, membrane preparations areprovided. The membrane preparations of the invention may typically beused for screening purposes, and may be obtained by standard techniques.

[0097] In a preferred embodiment, frozen intact cells of the inventionare homogenised while in cold water suspension and a membrane pellet iscollected after centrifugation. The pellet may then be washed in coldwater and dialysed to remove endogenous ligands that could compete forbinding in the assays. The dialysed membranes may be used as such, orafter storage in lyophilised form.

[0098] KCNQ5 Active Chemical Compounds

[0099] In another aspect the invention relates to chemical compoundscapable of binding to, and showing activity at potassium channelscontaining one or more KCNQ5 subunits. In the context of this inventionsuch compounds are termed KCNQ5 active compounds.

[0100] The KCNQ5 active compounds of the invention have therapeuticpotential, and may be used for the manufacture of pharmaceuticalcompositions.

[0101] The KCNQ5 active compounds of the invention may in particular beused in diagnosis, treatment, prevention or alleviation of diseasesrelated to diseases or adverse conditions of the CNS, includingaffective disorders, Alzheimer's disease, anxiety, ataxia, CNS damagecaused by trauma, stroke or neurodegenerative illness, cognitivedeficits, compulsive behaviour, dementia, depression, Huntington'sdisease, mania, memory impairment, memory disorders, memory dysfunction,motion. disorders, motor disorders, neurodegenerative diseases,Parkinson's disease and Parkinson-like motor disorders, phobias, Pick'sdisease, psychosis, schizophrenia, spinal cord damage, stroke, tremor,seizures, convulsions and epilepsy.

[0102] Currently two compounds have been identified. As a preferredembodiment the invention therefore provides1,3-dihydro-1-phenyl-3,3-bis-(4-pyridylmethyl)-2H-indol-2-one(Linopirdine) and 10,10-bis-(4-pyridinyl-methyl)-9-(10H)-antracenone(XE991) for use in the manufacture of a pharmaceutical composition forthe diagnosis, treatment, prevention or alleviation of the abovediseases.

[0103] Screening of Drugs

[0104] In a further aspect the invention provides methods for screeningfor KCNQ5 active compounds, i.e. chemical compounds capable of bindingto, and showing activity at potassium channels containing one or moreKCNQ5 subunits. The activity determined may be inhibitory activity,stimulating activity, or other modulatory activity.

[0105] In particular the KCNQ5 active compound may induce a secondmessenger response, which cause a change of the molecularcharacteristics of the cell, e.g. the ion flux, enzyme activation,changes in the level of intracellular Ca²⁺ or H⁺, changes cyclicnucleotides such as cAMP, cADP, cGMP, and cGDP, etc.

[0106] Therefore, in another aspect, the invention provides a method foridentifying functional ligands for a human potassium channel, comprisinga KCNQ5 subunit, which method comprises transfecting cells with one ormore polypeptides of the invention, encoding a KCNQ5 channel subunit,and detecting the effect on the signal transduction pathway caused inthese cells by binding of the ligands to the receptor by a reportersystem.

[0107] Such chemical compounds can be identified by one of, or bothmethods described below.

[0108] Binding Studies

[0109] Binding studies are usually carried out by subjecting the targetto binding with a labelled, selective agonist (binding agent), to form alabelled complex, followed by determination of the degree ofdisplacement caused by the test compound upon addition to the complex.

[0110] In a specific aspect the invention provides a method of screeninga chemical compound for capability of binding to a potassium channelcomprising at least one KCNQ5 channel subunit, which method comprisesthe steps of (i) subjecting a KCNQ5 channel subunit containing cell tothe action of a KCNQ5 binding agent to form a complex with the KCNQ5channel subunit containing cell; (ii) subjecting the complex of step (i)to the action of the chemical compound to be tested; and (iii) detectingthe displacement of the KCNQ5 binding agent from the complex with the.KCNQ5 channel subunit containing cell.

[0111] The KCNQ5 channel subunit containing cell preferably is a cell ofthe invention as described above.

[0112] The KCNQ5 binding agent preferably is a radioactively labelled1,3-dihydro-1-phenyl-3,3-bis-(4-pyridylmethyl)-2H-indol-2-one(Linopirdine); or 10,10-bis-(4-pyridinyl-methyl)-9-(10H)-antracenone(XE991).

[0113] In a even more preferred embodiment, the binding agent islabelled with ³H, and the displacement of the KCNQ5 binding agent fromthe complex with the KCNQ5 channel subunit containing cell is detectedby measuring the amount of radioactivity by conventional liquidscintillation counting.

[0114] Activity Studies

[0115] The KCNQ5 channel agonists may affect the potassium channel invarious ways. The agonist may in particular show inhibitory activity,stimulating activity, or other modulatory activity.

[0116] In a specific aspect the invention provides a method fordetermining the activity at potassium channels containing one or moreKCNQ5 subunits. According to this method a KCNQ5 channel subunitcontaining cell is subjecting to the action of the chemical compound tobe tested, and the activity is detected by way of monitoring themembrane potential, the current, the potassium flux, or the secondarycalcium influx of the KCNQ5 channel subunit containing cell, preferablya genetically manipulated as described above.

[0117] The membrane potential and the current may be monitored byelectrophysiologic methods, including patch clamp techniques, such ascurrent clamp technology and two-electrode voltage clamp technology, orby spectroscopic methods, such as fluorescence methods.

[0118] In a preferred embodiment, monitoring of the membrane potentialof the KCNQ5 channel subunit containing cell is performed by patch clamptechniques.

[0119] In another preferred embodiment, monitoring of the membranepotential of the KCNQ5 channel subunit containing cell is performed byspectroscopic methods, e.g. using fluorescence methods. In a morespecific embodiment, the KCNQ5 channel subunit containing cell is mixedwith a membrane potential indicating agent, that allow for adetermination of changes in the membrane potential of the cell, causedby the addition of the test compound. The membrane potential indicatingagent may in particular be a fluorescent indicator, preferablyDIBAC₄(3), DiOC5(3), and DiOC2(3).

[0120] In yet a preferred embodiment, monitoring of the membranepotential of the KCNQ5 channel subunit containing cell is performed byspectroscopic methods, e.g. using a FLIPR assay (Fluorescence ImagePlate Reader; available from Molecular Devices).

[0121] Screening of Genetic Material

[0122] In a further aspect the invention relates to the use of apolynucleotide sequence of the invention for the screening of geneticmaterials. By this method, individuals bearing a gene identical orhomologous to a polynucleotide of the invention may be identified.

[0123] In the screening method of the invention, a polynucleotide of theinvention, or any fragment or sub-sequence hereof, is employed. For theidentification of individuals bearing mutated genes preferably themutated forms of the polynucleotide represented by SEQ ID NO: 1 areemployed.

[0124] In the screening method of the invention only short sequencesneeds to be employed depending on the actual method used. For SSCA,several hundreds of base pairs may be needed, for oligonucleotide or PCRhybridisation only of from about 10 to about 50 basepairs may be needed.

[0125] The screening may be accomplished by conventional methods,including hybridisation, SSCA analysis, and microarray technology (DNAchip technology). The hybridisation protocol described above representsa suitable protocol for use in a screening method of the invention.

[0126] Transgenic Animals

[0127] Transgenic animal models provide the means, in vivo, to screenfor therapeutic compounds. Since KCNQ5 is expressed also in brain, theymay be helpful in screening for drugs effective in CNS disorders, e.g.epilepsy.

[0128] By transgene is meant any piece of polynucleotide which isinserted by artifice into a cell, and thus becomes part of the genome ofthe organism that develops from that cell. Such a transgene may includea gene which is partly or entirely heterologous (i.e. foreign) to thetransgenic organism, or it may represent a gene homologous to anendogenous gene of the organism.

[0129] By a transgenic animal is meant any organism holding a cell whichincludes a polynucleotide sequence which is inserted into that cell byartifice, and which cell becomes part of the transgenic organism whichdevelops from that cell. Such a transgene may be partly or entirelyheterologous to the transgenic animal. Although transgenic micerepresent a preferred embodiment of the invention, other transgenicmammals including, but not limited to transgenic rodents (e.g. hamsters,guinea pigs, rabbits and rats), and transgenic pigs, cattle, sheep andgoats may be created by standard techniques and are included in theinvention.

[0130] Preferably, the transgene is inserted by artifice into thenuclear genome.

[0131] Knock-Out and Knock-In Animals

[0132] The transgenic knock-out animal models may be developed byhomologous recombination of embryonic stem cells with constructscontaining genomic sequence from the KCNQ5 gene, that lead to a loss offunction of the gene after insertion into the endogenous gene.

[0133] By knock-out mutation is meant an alteration in thepolynucleotide sequence that reduces the biological activity of thepolypeptide normally encoded therefrom. In order to create a trueknock-out model, the biological activity of the expressed polypeptideshould be reduced by at least 80% relative to the un-mutated gene. Themutation may in particular be a substitution, an insertion, a deletion,a frameshift mutation, or a mis-sense mutation. Preferably the mutationis a substitution, an insertion or a deletion.

[0134] To further assess the role of KCNQ5 at an organism level, thegeneration of an animal, preferably a mouse, lacking the intact KCNQ5gene, or bearing a mutated KCNQ5 gene, is desired.

[0135] A replacement-type targeting vector, which may be used to createa knock-out model, may be constructed using an isogenic genomic clone,e.g. from a mouse strain such as 129/Sv (Stratagene Inc., La Jolla,Calif.). The targeting vector may be introduced into a suitably-derivedline of embryonic stem (ES) cells by electroporation to generate ES celllines that carry a profoundly truncated form of the KCNQ5 gene. Thetargeted cell lines may then be injected into a mouse blastula stageembryo to generate chimeric founder mice. Heterozygous offspring may beinterbred to homozygosity.

[0136] Animal models for over-expression may be generated by integratingone or more polynucleotide sequence of the invention into the genomeaccording to standard techniques.

[0137] The procedures disclosed herein involving the molecularmanipulation of nucleic acids are known to those skilled in the art, andare described by e.g. Fredrick M A et al. [Fredrick MA et al.: ShortProtocols in Molecular Biology; John Wiley and Sons, 1995] and Sambrooket al. [Sambrook et al.: Molecular Cloning: A Laboratory Manual; 2. Ed.,Cold Spring Harbor Lab.; Cold Spring Harbor, N.Y. 1989], and inAlexandra L J (Ed.): Gene Targeting: A practical approach; OxfordUniversity Press (Oxford, New York, Tokyo), 1993.

BRIEF DESCRIPTION OF THE DRAWINGS

[0138] The present invention is further illustrated by reference to theaccompanying drawing, in which:

[0139]FIG. 1 shows the electrophysiological properties of KCNQ5 currents[I/μA vs. time/seconds]. Two-electrode voltage-clamp current traces froma Xenopus oocyte injected with KCNQ5 cRNA. Starting from a holdingpotential of −80 mV, cells were clamped for 2 seconds to voltagesbetween −80 and +40 mV, in +10 mV steps, followed by a constant testpulse to −30 mV; and

[0140]FIG. 2 shows the electrophysiological properties of currentsarising from the co-expression of KCNQ5 with KCNQ3 [I/μA vs.time/seconds]. Starting from a holding potential of −80 mV cells wereclamped for 2 seconds to voltages between −80 and +40 mV, in +10 mVsteps, followed by a constant test pulse to −30 mV; and

[0141]FIG. 3 shows the electrophysiological properties of currentsarising from the co-expression of KCNQ5 with KCNQ4 (2B) [I/μA vs.time/seconds]. Starting from a holding potential of −80 mV cells wereclamped for 2 seconds to voltages between −80 and +40 mV, in +10 mVsteps, followed by a constant test pulse to −30 mV;

[0142] FIGS. 4A-G show the electrophysiological properties of KCNQ5.Both the splice variant I (found in brain) and splice variant III (foundin muscle) were expressed in Xenopus oocytes and examined bytwo-electrode voltage clamping. Both variants activate slowly upondepolarisation, but form I (4A) initially activates slower than themuscle form III (4B). Starting from a holding potential of −80 mV, thevoltage was stepped for 0.8 seconds to values between −100 and +40 mV insteps of 10 mV, followed by a voltage step to −30 mV (see inset, panelA). Channel activation by depolarization was fitted (for 2 sec. steps)by a sum of three exponential functions. For a step to +20 mV, the rateconstants were ô₁=37.2±2.2 ms, ô₂=246±17 ms, and ô₃=1112±91 ms forsplice variant I, while these constants were ô₁=24.5±0.8 ms, ô₂=163±6ms, and ô₃=1690±46 ms (±SEM, n=16). This difference in kinetics alsoresults in different curves when a typical M-current protocol is used(4C, variant I; 4D, variant III). Variant I induces currents thatkinetically resemble M-currents. In this protocol, the membrane voltageis clamped for 1 second to voltages between −30 and −90 mV in steps of−10 mV, from a holding potential of −30 mV. This was followed by a stepto −30 mV (panel C, inset). (4E, 4F), apparent open probabilities ofvariants I (4E) and variant III (4F) as a function of voltage obtainedfrom tail current analysis as described in Methods. Mean values obtainedfrom 12 oocytes are shown. Fitting a Boltzmann equation yieldedV_(1/2)=−46±1 mV and an apparent gating charge of z=2.8±0.1 for isoformI, and V_(1/2)=−48±1 mV and an apparent gating charge of z=2.5±0.1 forisoform III. For this fit, values at potentials more positive than +10mV were excluded, as these are probably affected by a second(inactivation) gating process which leads to a decrease of apparentp_(open) at more positive potentials. (4G), ion selectivity of KCNQ5currents (variant 1). Extracellular sodium was replaced by equimolaramounts of potassium. The reversal potential is shown as a function ofthe potassium concentration. This yielded a slope of 51 mV/decadepotassium concentration, indicating a highly selective potassiumchannel. Data are from 10 oocytes from 2 different batches;

[0143] FIGS. 5A-C show the pharmacology of KCNQ5 and KCNQ3/5 heteromers.(5A) Inhibition of KCNQ5 homomers by extracellular linopirdine (downtriangles), XE991 (up triangles) and TEA (squares). IC₅₀ values of 51±5μM, 65±4 μM and 71±17 mM, respectively, were obtained from the plottedfit curves. (5B) Niflumic acid alters the voltage dependence of theapparent p_(open) In the presence of 500 μM niflumic acid (opencircles), the voltage dependence is shifted about 20 mV towards negativepotentials. (5C) TEA sensitivity is altered in KCNQ3/5 heteromers.

[0144] Coexpression of KCNO5 and KCNQ3 (1:1) (diamonds) increased theIC₅₀ value to ⁻200 mM, coexpression with the KCNQ3 (T323Y) mutant(circles) decreased the IC₅₀ to ⁻30 mM. Data points in panels A and Care the means ±SEM of 4 to 12 individual measurements. p_(open) wasdetermined as in FIG. 3;

[0145]FIG. 6 shows the inhibition of KCNQ5 currents by stimulating M1receptors which were co-expressed in Xenopus oocytes:

[0146] (6A) currents before stimulating the M1 receptor;

[0147] (6B) current observed 3 minutes after applying 10 μM muscarine.The pulse protocol is shown in the inset of panel b. No effect of 10 μMmuscarine or oxotremorine methiodide was found in oocytes injected withKCNQ5 alone; and

[0148] FIGS. 7A-C show the interactions between KCNQ2 and KCNQ5 (7A, 7B)and KCNQ3 and KCNQ5 (7C):

[0149] (7A) currents at the end of a two second pulse to 0 mV from aholding potential of −80 mV of oocytes injected with differentcombinations of KCNQ cRNAs (always 10 ng total amount of RNA). Thecurrent amplitude elicited by co-injecting KCNQ2 and KCNQ5 could beexplained by a linear superposition of currents. Co-injection of KCNQ5with the dominant negative mutant KCNQ2(G279S) or of KCNQ2 with theequivalent mutant KCNQ5(G278S) lead to a roughly 50% reduction incurrent amplitude, which is consistent with a lack of interaction sinceonly 50% of WT cRNA was injected;

[0150] (7B) normalized current traces of experiments used for panel (A).Currents elicited by the depolarizing pulse to 0 mV are shown. KCNQ2(labeled Q2) activates faster than KCNQ5 (Q5), and the co-expression ofboth yielded currents that may be explained by a linear superposition.Co-injecting KCNQ2 with the dominant negative (and otherwisenon-functional) mutant KCNQ5(G278S) yielded currents that werekinetically similar to KCNQ2, and currents from a KCNQ5/KCNQ2(G279S)co-injection resembled KCNQ5 currents;

[0151] (7C), interactions between KCNQ3 and KCNQ5 measured as in panel(a). KCNQ3 yields only very small currents in Xenopus oocytes. Currentswere enlarged when KCNQ5 was co-expressed with small amounts of KCNQ3,and reduced when co-expressed with larger amounts. The dominant negativemutant KCNQ3(G318S) decreased currents significantly below 50% of WTKCNQ5 currents that would be expected from the injected 50% of WT KCNQ5cRNA in this experiment.

EXAMPLES

[0152] The invention is further illustrated with reference to thefollowing examples which are not intended to be in any way limiting tothe scope of the invention as claimed.

Example 1 Cloning and Characterisation of KCNQ5 cDNA

[0153] Using a near full-length KCNQ3 potassium channel cDNA as a probe,a human thalamus cDNA λGT11 phage library (Clontech, #HL5009b) wasscreened, and a partial cDNA clone encoding a protein fragmenthomologous to KCNQ potassium channels was isolated. It was distinct fromthe known members KCNQ1 (KvLQT1), KCNQ2, KCNQ3 and KCNQ4. We named thenovel gene KCNQ5. Overlapping cDNA's containing the entire open readingframe were obtained by re-screening the cDNA library and by extendingthe 5′ end in RACE (rapid amplification of cDNA ends) experiments usinga Marathon kit (Clontech) with human brain cDNA. A complete cDNA wasassembled and cloned into the oocyte expression vector PTLN [Lorenz C,Pusch M & Jentsch T J: Heteromultimeric CLC chloride channels with novelproperties; Proc. Natl. Acad. Sci. USA 1996 93 13362-13366].

[0154] The cDNA encodes a polypeptide of 897 amino acids with apredicted mass of 99 kDa (SEQ ID NO: 2). Its overall amino-acid identityto KCNQ1, KCNQ2, KCNQ3 and KCNQ4 is 43%, 58%, 54%, and 61% respectively.Together with these proteins, it forms a distinct branch of thesuperfamily of voltage-gated potassium channels. As a typical member ofthis gene family, KCNQ5 has 6 predicted transmembrane domains and aP-loop between transmembrane domains S5 and S6. In potassium channels,which are tetramers of identical or homologous subunits, four of thesehighly conserved P-loops combine to form the ion-selective pore. Asother KCNQ channels, KCNQ5 has a long predicted cytoplasmic carboxyterminus that accounts for about half of the protein. A conserved regionpresent in the carboxy termini of KCNQ1, -2, -3 and 4 is also present inKCNQ5.

[0155] The sequence of KCNQ5 predicts several potential sites forphosphorylation by protein kinase C and one for protein kinase A. Incontrast to KCNQ1 and KCNQ2, however, it lacks an amino terminalconsensus site for cAMP-dependent phosphorylation.

[0156] A human multiple tissue Northern blot (Clontech, #7760-1) wasprobed with a cDNA fragment of KCNQ5. The fragment was labelled with ³²Pusing the Rediprime labelling kit (Amersham). Hybridisation wasperformed in ExpressHyb solution according to the instructions of themanufacturer (Clontech). The filter was then exposed to Kodak BioMaxfilm for 4 days.

[0157] Northern analysis of KCNQ5 expression in human tissues revealed aband of ≈9 kb in brain.

Example 2 Functional Expression of KCNQ5 Potassium Channel Subunits

[0158] KCNQ5 was expressed in Xenopus oocytes and its activity wasinvestigated by two-electrode voltage clamping.

[0159] After linearization of the KCNQ5-containing pTLN vector withHpal, capped cRNA was transcribed in vitro using the mMessage mMachinecRNA synthesis kit (Ambion). Usually 5-15 ng of cRNA were injected intoXenopus oocytes previously isolated by manual defolliculation and shortcollagenase treatment. In co-expression experiments cRNAs were injectedat a 1:1 ratio. Oocytes were kept at 17° C. in modified Barth's solution(90 mM NaCl, 1 mM KCl, 0.41 mM CaCl₂, 0.33 mM Ca(NO₃)₂, 0.82 mM MgSO₄,10 mM HEPES, 100 U penicillin—100 μg streptomycin/ml, pH 7.6).

[0160] Standard two-electrode voltage-clamp measurements were performedat room temperature 2-4 days after injection using a Turbotec 05amplifier (npi instruments, Tamm, Germany) and pClamp 5.5 software (AxonInstruments). Currents were usually recorded in ND98 solution (see Table2). Linopirdine (RBI, Natick, Mass.) was prepared as a 100 mM stocksolution in DMSO and added to a final concentration of 200 μM to ND98.TABLE 2 Solution contents (Concentrations in mM) ND98 ND 100 KD100 Rb100Cs100  98 NaCl  100 NaCl  100 KCl  100 RbCl  100 CsCl   2 KCl 0.2 CaCl₂ 0.2 CaCl₂  0.2 CaCl₂  0.2 CaCl₂  0.2 CaCl₂ 2.8 MgCl₂  2.8 MgCl₂  2.8MgCl₂  2.8 MgCl₂  2.8 MgCl₂ 5 mM HEPES, pH 7.4

[0161] To determine the voltage dependence of apparent open probability,oocytes were clamped for 2 seconds to values between −80 mV to +40 mV,in 10 mV steps, followed by a constant −30 mV test pulse. Tail currentsextrapolated to t=0 were obtained from a mono-exponential fit,normalised to the value at 0 mV and used for the analysis of apparentp_(open). Data analysis used PClamp6 and Microcal Origin 5.0.

[0162] Similar to KCNQ1, KCNQ2, KCNQ3, and KCNQ4 also KCNQ5 yieldedcurrents that activated upon depolarisation (FIG. 1). Compared to KCNQ1,KCNQ2 and KCNQ2/3 channels, however, current activation was slower andoccurred with a time constant in the order of 600 ms at +20 mV(KCNQ2/KCNQ3 channels have a corresponding time constant of ≈300 ms).Deactivation of currents at physiological resting potentials (≈−30 mV)was considerably faster (FIG. 1). Similar to KCNQ2, macroscopic currentsoften showed some inward rectification at positive potentials. KCNQ5currents were inhibited by more than 80% by 5 mM Ba⁺⁺.

[0163] KCNQ1 assembles with mink (also known as KCNE1 or IsK) to formchannels that yield larger currents and activate much slower. Wetherefore tested by co-expression whether mink affects KCNQ5 as well. Atconcentrations (1 ng minK cRNA per oocyte) leading to drastic changes inKCNQ1 currents in parallel experiments, there was just a slight changein KCNQ5 currents.

[0164] Different KCNQ subunits can form heteromeric channels.Co-expression of KCNQ2 with KCNQ3, but not with KCNQ1, gave currentsthat were about tenfold larger than those from homomeric channels. Sincealso KCNQ2, KCNQ3 and KCNQ4 are expressed in the brain, we investigatedwhether these proteins interact functionally. Oocytes co-injected (atthe same total cRNA concentration) with KCNQ2 and KCNQ5 cRNAs yieldedcurrents that seemed not different from a linear superposition ofcurrents from the respective homomeric channels.

[0165] By contrast, co-expression of KCNQ3 with KCNQ5 yielded currentsthat were significantly larger than could be explained by asuperposition of currents from the respective homomeric channels (FIGS.2A). Further, co-expression of KCNQ5 and KCNQ4 decreased currents whencompared to homomeric channels (FIG. 2B).

[0166] Linopirdine, a potent and rather specific inhibitor forM-currents, nearly completely inhibits KCNQ2/KCNQ3 channels at aconcentration of 200 μM.

[0167] This concentration of Linopirdine inhibited KCNQ5 by about 80%.

1 10 1 3137 DNA Homo sapiens CDS (1)..(2691) 1 atg aag gat gtg gag tcgggc cgg ggc agg gtg ctg ctg aac tcg gca 48 Met Lys Asp Val Glu Ser GlyArg Gly Arg Val Leu Leu Asn Ser Ala 1 5 10 15 gcc gcc agg ggc gac ggcctg cta ctg ctg ggc acc cgc gcg gcc acg 96 Ala Ala Arg Gly Asp Gly LeuLeu Leu Leu Gly Thr Arg Ala Ala Thr 20 25 30 ctc ggt ggc ggc ggc ggt ggcctg agg gag agc cgc cgg ggc aag cag 144 Leu Gly Gly Gly Gly Gly Gly LeuArg Glu Ser Arg Arg Gly Lys Gln 35 40 45 ggg gcc cgg atg agc ctg ctg gggaag ccg ctc tct tac acg agt agc 192 Gly Ala Arg Met Ser Leu Leu Gly LysPro Leu Ser Tyr Thr Ser Ser 50 55 60 cag agc tgc cgg cgc aac gtc aag taccgg cgg gtg cag aac tac ctg 240 Gln Ser Cys Arg Arg Asn Val Lys Tyr ArgArg Val Gln Asn Tyr Leu 65 70 75 80 tac aac gtg ctg gag aga ccc cgc ggctgg gcg ttc atc tac cac gct 288 Tyr Asn Val Leu Glu Arg Pro Arg Gly TrpAla Phe Ile Tyr His Ala 85 90 95 ttc gtt ttt ctc ctt gtc ttt ggt tgc ttgatt ttg tca gtg ttt tct 336 Phe Val Phe Leu Leu Val Phe Gly Cys Leu IleLeu Ser Val Phe Ser 100 105 110 acc atc cct gag cac aca aaa ttg gcc tcaagt tgc ctc ttg atc ctg 384 Thr Ile Pro Glu His Thr Lys Leu Ala Ser SerCys Leu Leu Ile Leu 115 120 125 gag ttc gtg atg att gtc gtc ttt ggt ttggag ttc atc att cga atc 432 Glu Phe Val Met Ile Val Val Phe Gly Leu GluPhe Ile Ile Arg Ile 130 135 140 tgg tct gcg ggt tgc tgt tgt cga tat agagga tgg caa gga aga ctg 480 Trp Ser Ala Gly Cys Cys Cys Arg Tyr Arg GlyTrp Gln Gly Arg Leu 145 150 155 160 agg ttt gct cga aag ccc ttc tgt gttata gat acc att gtt ctt atc 528 Arg Phe Ala Arg Lys Pro Phe Cys Val IleAsp Thr Ile Val Leu Ile 165 170 175 gct tca ata gca gtt gtt tct gca aaaact cag ggt aat att ttt gcc 576 Ala Ser Ile Ala Val Val Ser Ala Lys ThrGln Gly Asn Ile Phe Ala 180 185 190 acg tct gca ctc aga agt ctc cgt ttccta cag atc ctc cgc atg gtg 624 Thr Ser Ala Leu Arg Ser Leu Arg Phe LeuGln Ile Leu Arg Met Val 195 200 205 cgc atg gac cga agg gga ggc act tggaaa tta ctg ggt tca gtg gtt 672 Arg Met Asp Arg Arg Gly Gly Thr Trp LysLeu Leu Gly Ser Val Val 210 215 220 tat gct cac agc aag gaa tta atc acagct tgg tac ata gga ttt ttg 720 Tyr Ala His Ser Lys Glu Leu Ile Thr AlaTrp Tyr Ile Gly Phe Leu 225 230 235 240 gtt ctt att ttt tcg tct ttc cttgtc tat ctg gtg gaa aag gat gcc 768 Val Leu Ile Phe Ser Ser Phe Leu ValTyr Leu Val Glu Lys Asp Ala 245 250 255 aat aaa gag ttt tct aca tat gcagat gct ctc tgg tgg ggc aca att 816 Asn Lys Glu Phe Ser Thr Tyr Ala AspAla Leu Trp Trp Gly Thr Ile 260 265 270 aca ttg aca act att ggc tat ggagac aaa act ccc cta act tgg ctg 864 Thr Leu Thr Thr Ile Gly Tyr Gly AspLys Thr Pro Leu Thr Trp Leu 275 280 285 gga aga ttg ctt tct gca ggc tttgca ctc ctt ggc att tct ttc ttt 912 Gly Arg Leu Leu Ser Ala Gly Phe AlaLeu Leu Gly Ile Ser Phe Phe 290 295 300 gca ctt cct gcc ggc att ctt ggctca ggt ttt gca tta aaa gta caa 960 Ala Leu Pro Ala Gly Ile Leu Gly SerGly Phe Ala Leu Lys Val Gln 305 310 315 320 gaa caa cac cgc cag aaa cacttt gag aaa aga agg aac cca gct gcc 1008 Glu Gln His Arg Gln Lys His PheGlu Lys Arg Arg Asn Pro Ala Ala 325 330 335 aac ctc att cag tgt gtt tggcgt agt tac gca gct gat gag aaa tct 1056 Asn Leu Ile Gln Cys Val Trp ArgSer Tyr Ala Ala Asp Glu Lys Ser 340 345 350 gtt tcc att gca acc tgg aagcca cac ttg aag gcc ttg cac acc tgc 1104 Val Ser Ile Ala Thr Trp Lys ProHis Leu Lys Ala Leu His Thr Cys 355 360 365 agc cct acc aag aaa gaa caaggg gaa gca tca agc agt cag aag cta 1152 Ser Pro Thr Lys Lys Glu Gln GlyGlu Ala Ser Ser Ser Gln Lys Leu 370 375 380 agt ttt aag gag cga gtg cgcatg gct agc ccc agg ggc cag agt att 1200 Ser Phe Lys Glu Arg Val Arg MetAla Ser Pro Arg Gly Gln Ser Ile 385 390 395 400 aag agc cga caa gcc tcagta ggt gac agg agg tcc cca agc acc gac 1248 Lys Ser Arg Gln Ala Ser ValGly Asp Arg Arg Ser Pro Ser Thr Asp 405 410 415 atc aca gcc gag ggc agtccc acc aaa gtg cag aag agc tgg agc ttc 1296 Ile Thr Ala Glu Gly Ser ProThr Lys Val Gln Lys Ser Trp Ser Phe 420 425 430 aac gac cga acc cgc ttccgg ccc tcg ctg cgc ctc aaa agt tct cag 1344 Asn Asp Arg Thr Arg Phe ArgPro Ser Leu Arg Leu Lys Ser Ser Gln 435 440 445 cca aaa cca gtg ata gatgct gac aca gcc ctt ggc act gat gat gta 1392 Pro Lys Pro Val Ile Asp AlaAsp Thr Ala Leu Gly Thr Asp Asp Val 450 455 460 tat gat gaa aaa gga tgccag tgt gat gta tca gtg gaa gac ctc acc 1440 Tyr Asp Glu Lys Gly Cys GlnCys Asp Val Ser Val Glu Asp Leu Thr 465 470 475 480 cca cca ctt aaa actgtc att cga gct atc aga att atg aaa ttt cat 1488 Pro Pro Leu Lys Thr ValIle Arg Ala Ile Arg Ile Met Lys Phe His 485 490 495 gtt gca aaa cgg aagttt aag gaa aca tta cgt cca tat gat gta aaa 1536 Val Ala Lys Arg Lys PheLys Glu Thr Leu Arg Pro Tyr Asp Val Lys 500 505 510 gat gtc att gaa caatat tct gct ggt cat ctg gac atg ttg tgt aga 1584 Asp Val Ile Glu Gln TyrSer Ala Gly His Leu Asp Met Leu Cys Arg 515 520 525 att aaa agc ctt caaaca cgt gtt gat caa att ctt gga aaa ggg caa 1632 Ile Lys Ser Leu Gln ThrArg Val Asp Gln Ile Leu Gly Lys Gly Gln 530 535 540 atc aca tca gat aagaag agc cga gag aaa ata aca gca gaa cat gag 1680 Ile Thr Ser Asp Lys LysSer Arg Glu Lys Ile Thr Ala Glu His Glu 545 550 555 560 acc aca gac gatctc agt atg ctc ggt cgg gtg gtc aag gtt gaa aaa 1728 Thr Thr Asp Asp LeuSer Met Leu Gly Arg Val Val Lys Val Glu Lys 565 570 575 cag gta cag tccata gaa tcc aag ctg gac tgc cta cta gac atc tat 1776 Gln Val Gln Ser IleGlu Ser Lys Leu Asp Cys Leu Leu Asp Ile Tyr 580 585 590 caa cag gtc cttcgg aaa ggc tct gcc tca gcc ctc gct ttg gct tca 1824 Gln Gln Val Leu ArgLys Gly Ser Ala Ser Ala Leu Ala Leu Ala Ser 595 600 605 ttc cag atc ccacct ttt gaa tgt gaa cag aca tct gac tat caa agc 1872 Phe Gln Ile Pro ProPhe Glu Cys Glu Gln Thr Ser Asp Tyr Gln Ser 610 615 620 cct gtg gat agcaaa gat ctt tcg ggt tcc gca caa aac agt ggc tgc 1920 Pro Val Asp Ser LysAsp Leu Ser Gly Ser Ala Gln Asn Ser Gly Cys 625 630 635 640 tta tcc agatca act agt gcc aac atc tcg aga ggc ctg cag ttc att 1968 Leu Ser Arg SerThr Ser Ala Asn Ile Ser Arg Gly Leu Gln Phe Ile 645 650 655 ctg acg ccaaat gag ttc agt gcc cag act ttc tac gcg ctt agc cct 2016 Leu Thr Pro AsnGlu Phe Ser Ala Gln Thr Phe Tyr Ala Leu Ser Pro 660 665 670 act atg cacagt caa gca aca cag gtg cca att agt caa agc gat ggc 2064 Thr Met His SerGln Ala Thr Gln Val Pro Ile Ser Gln Ser Asp Gly 675 680 685 tca gca gtggca gcc acc aac acc att gca aac caa ata aat acg gca 2112 Ser Ala Val AlaAla Thr Asn Thr Ile Ala Asn Gln Ile Asn Thr Ala 690 695 700 ccc aag ccagca gcc cca aca act tta cag atc cca cct cct ctc cca 2160 Pro Lys Pro AlaAla Pro Thr Thr Leu Gln Ile Pro Pro Pro Leu Pro 705 710 715 720 gcc atcaag cat ctg ccc agg cca gaa act ctg cac cct aac cct gca 2208 Ala Ile LysHis Leu Pro Arg Pro Glu Thr Leu His Pro Asn Pro Ala 725 730 735 ggc ttacag gaa agc att tct gac gtc acc acc tgc ctt gtt gcc tcc 2256 Gly Leu GlnGlu Ser Ile Ser Asp Val Thr Thr Cys Leu Val Ala Ser 740 745 750 aag gaaaat gtt cag gtt gca cag tca aat ctc acc aag gac cgt tct 2304 Lys Glu AsnVal Gln Val Ala Gln Ser Asn Leu Thr Lys Asp Arg Ser 755 760 765 atg aggaaa agc ttt gac atg gga gga gaa act ctg ttg tct gtc tgt 2352 Met Arg LysSer Phe Asp Met Gly Gly Glu Thr Leu Leu Ser Val Cys 770 775 780 ccc atggtg ccg aag gac ttg ggc aaa tct ttg tct gtg caa aac ctg 2400 Pro Met ValPro Lys Asp Leu Gly Lys Ser Leu Ser Val Gln Asn Leu 785 790 795 800 atcagg tcg acc gag gaa ctg aat ata caa ctt tca ggg agt gag tca 2448 Ile ArgSer Thr Glu Glu Leu Asn Ile Gln Leu Ser Gly Ser Glu Ser 805 810 815 agtggc tcc aga ggc agc caa gat ttt tac ccc aaa tgg agg gaa tcc 2496 Ser GlySer Arg Gly Ser Gln Asp Phe Tyr Pro Lys Trp Arg Glu Ser 820 825 830 aaattg ttt ata act gat gaa gag gtg ggt ccc gaa gag aca gag aca 2544 Lys LeuPhe Ile Thr Asp Glu Glu Val Gly Pro Glu Glu Thr Glu Thr 835 840 845 gacact ttt gat gcc gca ccg cag cct gcc agg gaa gct gcc ttt gca 2592 Asp ThrPhe Asp Ala Ala Pro Gln Pro Ala Arg Glu Ala Ala Phe Ala 850 855 860 tcagac tct cta agg act gga agg tca cga tca tct cag agc att tgt 2640 Ser AspSer Leu Arg Thr Gly Arg Ser Arg Ser Ser Gln Ser Ile Cys 865 870 875 880aag gca gga gaa agt aca gat gcc ctc agc ttg cct cat gtc aaa ctg 2688 LysAla Gly Glu Ser Thr Asp Ala Leu Ser Leu Pro His Val Lys Leu 885 890 895aaa taagttcttc attttctttc caggcatagc agttctttag ccatacatat 2741 Lyscattgcatga actatttcga aagcccttct aaaaagttga aattgcaaga atcgggaaga 2801acatgaaagg cagtttataa gcccgttacc ttttaattgc atgaaaatgc atgtttaggg 2861atggctaaaa ttccaaggtg catcgacatt aacccactca tttagtaatg taccttgagt 2921taaaaagcct gagaaaccaa acacagctaa tgctatgggg tgtatgaata tgtcaagttt 2981aggtcattta gaagatttga cactgtattt tgaaattatg ggagtaaaca ccttcaaatt 3041tcaggcattt ctgctttgtg actaaataca aactacattt tcaagattag gccataatgt 3101atatttaaac acaatggcta tcaacagctg ctaata 3137 2 897 PRT Homo sapiens 2Met Lys Asp Val Glu Ser Gly Arg Gly Arg Val Leu Leu Asn Ser Ala 1 5 1015 Ala Ala Arg Gly Asp Gly Leu Leu Leu Leu Gly Thr Arg Ala Ala Thr 20 2530 Leu Gly Gly Gly Gly Gly Gly Leu Arg Glu Ser Arg Arg Gly Lys Gln 35 4045 Gly Ala Arg Met Ser Leu Leu Gly Lys Pro Leu Ser Tyr Thr Ser Ser 50 5560 Gln Ser Cys Arg Arg Asn Val Lys Tyr Arg Arg Val Gln Asn Tyr Leu 65 7075 80 Tyr Asn Val Leu Glu Arg Pro Arg Gly Trp Ala Phe Ile Tyr His Ala 8590 95 Phe Val Phe Leu Leu Val Phe Gly Cys Leu Ile Leu Ser Val Phe Ser100 105 110 Thr Ile Pro Glu His Thr Lys Leu Ala Ser Ser Cys Leu Leu IleLeu 115 120 125 Glu Phe Val Met Ile Val Val Phe Gly Leu Glu Phe Ile IleArg Ile 130 135 140 Trp Ser Ala Gly Cys Cys Cys Arg Tyr Arg Gly Trp GlnGly Arg Leu 145 150 155 160 Arg Phe Ala Arg Lys Pro Phe Cys Val Ile AspThr Ile Val Leu Ile 165 170 175 Ala Ser Ile Ala Val Val Ser Ala Lys ThrGln Gly Asn Ile Phe Ala 180 185 190 Thr Ser Ala Leu Arg Ser Leu Arg PheLeu Gln Ile Leu Arg Met Val 195 200 205 Arg Met Asp Arg Arg Gly Gly ThrTrp Lys Leu Leu Gly Ser Val Val 210 215 220 Tyr Ala His Ser Lys Glu LeuIle Thr Ala Trp Tyr Ile Gly Phe Leu 225 230 235 240 Val Leu Ile Phe SerSer Phe Leu Val Tyr Leu Val Glu Lys Asp Ala 245 250 255 Asn Lys Glu PheSer Thr Tyr Ala Asp Ala Leu Trp Trp Gly Thr Ile 260 265 270 Thr Leu ThrThr Ile Gly Tyr Gly Asp Lys Thr Pro Leu Thr Trp Leu 275 280 285 Gly ArgLeu Leu Ser Ala Gly Phe Ala Leu Leu Gly Ile Ser Phe Phe 290 295 300 AlaLeu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala Leu Lys Val Gln 305 310 315320 Glu Gln His Arg Gln Lys His Phe Glu Lys Arg Arg Asn Pro Ala Ala 325330 335 Asn Leu Ile Gln Cys Val Trp Arg Ser Tyr Ala Ala Asp Glu Lys Ser340 345 350 Val Ser Ile Ala Thr Trp Lys Pro His Leu Lys Ala Leu His ThrCys 355 360 365 Ser Pro Thr Lys Lys Glu Gln Gly Glu Ala Ser Ser Ser GlnLys Leu 370 375 380 Ser Phe Lys Glu Arg Val Arg Met Ala Ser Pro Arg GlyGln Ser Ile 385 390 395 400 Lys Ser Arg Gln Ala Ser Val Gly Asp Arg ArgSer Pro Ser Thr Asp 405 410 415 Ile Thr Ala Glu Gly Ser Pro Thr Lys ValGln Lys Ser Trp Ser Phe 420 425 430 Asn Asp Arg Thr Arg Phe Arg Pro SerLeu Arg Leu Lys Ser Ser Gln 435 440 445 Pro Lys Pro Val Ile Asp Ala AspThr Ala Leu Gly Thr Asp Asp Val 450 455 460 Tyr Asp Glu Lys Gly Cys GlnCys Asp Val Ser Val Glu Asp Leu Thr 465 470 475 480 Pro Pro Leu Lys ThrVal Ile Arg Ala Ile Arg Ile Met Lys Phe His 485 490 495 Val Ala Lys ArgLys Phe Lys Glu Thr Leu Arg Pro Tyr Asp Val Lys 500 505 510 Asp Val IleGlu Gln Tyr Ser Ala Gly His Leu Asp Met Leu Cys Arg 515 520 525 Ile LysSer Leu Gln Thr Arg Val Asp Gln Ile Leu Gly Lys Gly Gln 530 535 540 IleThr Ser Asp Lys Lys Ser Arg Glu Lys Ile Thr Ala Glu His Glu 545 550 555560 Thr Thr Asp Asp Leu Ser Met Leu Gly Arg Val Val Lys Val Glu Lys 565570 575 Gln Val Gln Ser Ile Glu Ser Lys Leu Asp Cys Leu Leu Asp Ile Tyr580 585 590 Gln Gln Val Leu Arg Lys Gly Ser Ala Ser Ala Leu Ala Leu AlaSer 595 600 605 Phe Gln Ile Pro Pro Phe Glu Cys Glu Gln Thr Ser Asp TyrGln Ser 610 615 620 Pro Val Asp Ser Lys Asp Leu Ser Gly Ser Ala Gln AsnSer Gly Cys 625 630 635 640 Leu Ser Arg Ser Thr Ser Ala Asn Ile Ser ArgGly Leu Gln Phe Ile 645 650 655 Leu Thr Pro Asn Glu Phe Ser Ala Gln ThrPhe Tyr Ala Leu Ser Pro 660 665 670 Thr Met His Ser Gln Ala Thr Gln ValPro Ile Ser Gln Ser Asp Gly 675 680 685 Ser Ala Val Ala Ala Thr Asn ThrIle Ala Asn Gln Ile Asn Thr Ala 690 695 700 Pro Lys Pro Ala Ala Pro ThrThr Leu Gln Ile Pro Pro Pro Leu Pro 705 710 715 720 Ala Ile Lys His LeuPro Arg Pro Glu Thr Leu His Pro Asn Pro Ala 725 730 735 Gly Leu Gln GluSer Ile Ser Asp Val Thr Thr Cys Leu Val Ala Ser 740 745 750 Lys Glu AsnVal Gln Val Ala Gln Ser Asn Leu Thr Lys Asp Arg Ser 755 760 765 Met ArgLys Ser Phe Asp Met Gly Gly Glu Thr Leu Leu Ser Val Cys 770 775 780 ProMet Val Pro Lys Asp Leu Gly Lys Ser Leu Ser Val Gln Asn Leu 785 790 795800 Ile Arg Ser Thr Glu Glu Leu Asn Ile Gln Leu Ser Gly Ser Glu Ser 805810 815 Ser Gly Ser Arg Gly Ser Gln Asp Phe Tyr Pro Lys Trp Arg Glu Ser820 825 830 Lys Leu Phe Ile Thr Asp Glu Glu Val Gly Pro Glu Glu Thr GluThr 835 840 845 Asp Thr Phe Asp Ala Ala Pro Gln Pro Ala Arg Glu Ala AlaPhe Ala 850 855 860 Ser Asp Ser Leu Arg Thr Gly Arg Ser Arg Ser Ser GlnSer Ile Cys 865 870 875 880 Lys Ala Gly Glu Ser Thr Asp Ala Leu Ser LeuPro His Val Lys Leu 885 890 895 Lys 3 676 PRT Homo sapiens 3 Met Ala AlaAla Ser Ser Pro Pro Arg Ala Glu Arg Lys Arg Trp Gly 1 5 10 15 Trp GlyArg Leu Pro Gly Ala Arg Arg Gly Ser Ala Gly Leu Ala Lys 20 25 30 Lys CysPro Phe Ser Leu Glu Leu Ala Glu Gly Gly Pro Ala Gly Gly 35 40 45 Ala LeuTyr Ala Pro Ile Ala Pro Gly Ala Pro Gly Pro Ala Pro Pro 50 55 60 Ala SerPro Ala Ala Pro Ala Ala Pro Pro Val Ala Ser Asp Leu Gly 65 70 75 80 ProArg Pro Pro Val Ser Leu Asp Pro Arg Val Ser Ile Tyr Ser Thr 85 90 95 ArgArg Pro Val Leu Ala Arg Thr His Val Gln Gly Arg Val Tyr Asn 100 105 110Phe Leu Glu Arg Pro Thr Gly Trp Lys Cys Phe Val Tyr His Phe Ala 115 120125 Val Phe Leu Ile Val Leu Val Cys Leu Ile Phe Ser Val Leu Ser Thr 130135 140 Ile Glu Gln Tyr Ala Ala Leu Ala Thr Gly Thr Leu Phe Trp Met Glu145 150 155 160 Ile Val Leu Val Val Phe Phe Gly Thr Glu Tyr Val Val ArgLeu Trp 165 170 175 Ser Ala Gly Cys Arg Ser Lys Tyr Val Gly Leu Trp GlyArg Leu Arg 180 185 190 Phe Ala Arg Lys Pro Ile Ser Ile Ile Asp Leu IleVal Val Val Ala 195 200 205 Ser Met Val Val Leu Cys Val Gly Ser Lys GlyGln Val Phe Ala Thr 210 215 220 Ser Ala Ile Arg Gly Ile Arg Phe Leu GlnIle Leu Arg Met Leu His 225 230 235 240 Val Asp Arg Gln Gly Gly Thr TrpArg Leu Leu Gly Ser Val Val Phe 245 250 255 Ile His Arg Gln Glu Leu IleThr Thr Leu Tyr Ile Gly Phe Leu Gly 260 265 270 Leu Ile Phe Ser Ser TyrPhe Val Tyr Leu Ala Glu Lys Asp Ala Val 275 280 285 Asn Glu Ser Gly ArgVal Glu Phe Gly Ser Tyr Ala Asp Ala Leu Trp 290 295 300 Trp Gly Val ValThr Val Thr Thr Ile Gly Tyr Gly Asp Lys Val Pro 305 310 315 320 Gln ThrTrp Val Gly Lys Thr Ile Ala Ser Cys Phe Ser Val Phe Ala 325 330 335 IleSer Phe Phe Ala Leu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala 340 345 350Leu Lys Val Gln Gln Lys Gln Arg Gln Lys His Phe Asn Arg Gln Ile 355 360365 Pro Ala Ala Ala Ser Leu Ile Gln Thr Ala Trp Arg Cys Tyr Ala Ala 370375 380 Glu Asn Pro Asp Ser Ser Thr Trp Lys Ile Tyr Ile Arg Lys Ala Pro385 390 395 400 Arg Ser His Thr Leu Leu Ser Pro Ser Pro Lys Pro Lys LysSer Val 405 410 415 Val Val Lys Lys Lys Lys Phe Lys Leu Asp Lys Asp AsnGly Val Thr 420 425 430 Pro Gly Glu Lys Met Leu Thr Val Pro His Ile ThrCys Asp Pro Pro 435 440 445 Glu Glu Arg Arg Leu Asp His Phe Ser Val AspGly Tyr Asp Ser Ser 450 455 460 Val Arg Lys Ser Pro Thr Leu Leu Glu ValSer Met Pro His Phe Met 465 470 475 480 Arg Thr Asn Ser Phe Ala Glu AspLeu Asp Leu Glu Gly Glu Thr Leu 485 490 495 Leu Thr Pro Ile Thr His IleSer Gln Leu Arg Glu His His Arg Ala 500 505 510 Thr Ile Lys Val Ile ArgArg Met Gln Tyr Phe Val Ala Lys Lys Lys 515 520 525 Phe Gln Gln Ala ArgLys Pro Tyr Asp Val Arg Asp Val Ile Glu Gln 530 535 540 Tyr Ser Gln GlyHis Leu Asn Leu Met Val Arg Ile Lys Glu Leu Gln 545 550 555 560 Arg ArgLeu Asp Gln Ser Ile Gly Lys Pro Ser Leu Phe Ile Ser Val 565 570 575 SerGlu Lys Ser Lys Asp Arg Gly Ser Asn Thr Ile Gly Ala Arg Leu 580 585 590Asn Arg Val Glu Asp Lys Val Thr Gln Leu Asp Gln Arg Leu Ala Leu 595 600605 Ile Thr Asp Met Leu His Gln Leu Leu Ser Leu His Gly Gly Ser Thr 610615 620 Pro Gly Ser Gly Gly Pro Pro Arg Glu Gly Gly Ala His Ile Thr Gln625 630 635 640 Pro Cys Gly Ser Gly Gly Ser Val Asp Pro Glu Leu Phe LeuPro Ser 645 650 655 Asn Thr Leu Pro Thr Tyr Glu Gln Leu Thr Val Pro ArgArg Gly Pro 660 665 670 Asp Glu Gly Ser 675 4 844 PRT Homo sapiens 4 MetVal Gln Lys Ser Arg Asn Gly Gly Val Tyr Pro Gly Pro Ser Gly 1 5 10 15Glu Lys Lys Leu Lys Val Gly Phe Val Gly Leu Asp Pro Gly Ala Pro 20 25 30Asp Ser Thr Arg Asp Gly Ala Leu Leu Ile Ala Gly Ser Glu Ala Pro 35 40 45Lys Arg Gly Ser Ile Leu Ser Lys Pro Arg Ala Gly Gly Ala Gly Ala 50 55 60Gly Lys Pro Pro Lys Arg Asn Ala Phe Tyr Arg Lys Leu Gln Asn Phe 65 70 7580 Leu Tyr Asn Val Leu Glu Arg Pro Arg Gly Trp Ala Phe Ile Tyr His 85 9095 Ala Tyr Val Phe Leu Leu Val Phe Ser Cys Leu Val Leu Ser Val Phe 100105 110 Ser Thr Ile Lys Glu Tyr Glu Lys Ser Ser Glu Gly Ala Leu Tyr Ile115 120 125 Leu Glu Ile Val Thr Ile Val Val Phe Gly Val Glu Tyr Phe ValArg 130 135 140 Ile Trp Ala Ala Gly Cys Cys Cys Arg Tyr Arg Gly Trp ArgGly Arg 145 150 155 160 Leu Lys Phe Ala Arg Lys Pro Phe Cys Val Ile AspIle Met Val Leu 165 170 175 Ile Ala Ser Ile Ala Val Leu Ala Ala Gly SerGln Gly Asn Val Phe 180 185 190 Ala Thr Ser Ala Leu Arg Ser Leu Arg PheLeu Gln Ile Leu Arg Met 195 200 205 Ile Arg Met Asp Arg Arg Gly Gly ThrTrp Lys Leu Leu Gly Ser Val 210 215 220 Val Tyr Ala His Ser Lys Glu LeuVal Thr Ala Trp Tyr Ile Gly Phe 225 230 235 240 Leu Cys Leu Ile Leu AlaSer Phe Leu Val Tyr Leu Ala Glu Lys Gly 245 250 255 Glu Asn Asp His PheAsp Thr Tyr Ala Asp Ala Leu Trp Trp Gly Leu 260 265 270 Ile Thr Leu ThrThr Ile Gly Tyr Gly Asp Lys Tyr Pro Gln Thr Trp 275 280 285 Asn Gly ArgLeu Leu Ala Ala Thr Phe Thr Leu Ile Gly Val Ser Phe 290 295 300 Phe AlaLeu Pro Ala Gly Ile Leu Gly Ser Gly Phe Ala Leu Lys Val 305 310 315 320Gln Glu Gln His Arg Gln Lys His Phe Glu Lys Arg Arg Asn Pro Ala 325 330335 Ala Gly Leu Ile Gln Ser Ala Trp Arg Phe Tyr Ala Thr Asn Leu Ser 340345 350 Arg Thr Asp Leu His Ser Thr Trp Gln Tyr Tyr Glu Arg Thr Val Thr355 360 365 Val Pro Met Tyr Arg Leu Ile Pro Pro Leu Asn Gln Leu Glu LeuLeu 370 375 380 Arg Asn Leu Lys Ser Lys Ser Gly Leu Ala Phe Arg Lys AspPro Pro 385 390 395 400 Pro Glu Pro Ser Pro Ser Gln Lys Val Ser Leu LysAsp Arg Val Phe 405 410 415 Ser Ser Pro Arg Gly Val Ala Ala Lys Gly LysGly Ser Pro Gln Ala 420 425 430 Gln Thr Val Arg Arg Ser Pro Ser Ala AspGln Ser Leu Glu Asp Ser 435 440 445 Pro Ser Lys Val Pro Lys Ser Trp SerPhe Gly Asp Arg Ser Arg Ala 450 455 460 Arg Gln Ala Phe Arg Ile Lys GlyAla Ala Ser Arg Gln Asn Ser Glu 465 470 475 480 Glu Ala Ser Leu Pro GlyGlu Asp Ile Val Asp Asp Lys Ser Cys Pro 485 490 495 Cys Glu Phe Val ThrGlu Asp Leu Thr Pro Gly Leu Lys Val Ser Ile 500 505 510 Arg Ala Val CysVal Met Arg Phe Leu Val Ser Lys Arg Lys Phe Lys 515 520 525 Glu Ser LeuArg Pro Tyr Asp Val Met Asp Val Ile Glu Gln Tyr Ser 530 535 540 Ala GlyHis Leu Asp Met Leu Ser Arg Ile Lys Ser Leu Gln Ser Arg 545 550 555 560Val Asp Gln Ile Val Gly Arg Gly Pro Ala Ile Thr Asp Lys Asp Arg 565 570575 Thr Lys Gly Pro Ala Glu Ala Glu Leu Pro Glu Asp Pro Ser Met Met 580585 590 Gly Arg Leu Gly Lys Val Glu Lys Gln Val Leu Ser Met Glu Lys Lys595 600 605 Leu Asp Phe Leu Val Asn Ile Tyr Met Gln Arg Met Gly Ile ProPro 610 615 620 Thr Glu Thr Glu Ala Tyr Phe Gly Ala Lys Glu Pro Glu ProAla Pro 625 630 635 640 Pro Tyr His Ser Pro Glu Asp Ser Arg Glu His ValAsp Arg His Gly 645 650 655 Cys Ile Val Lys Ile Val Arg Ser Ser Ser SerThr Gly Gln Lys Asn 660 665 670 Phe Ser Ala Pro Pro Ala Ala Pro Pro ValGln Cys Pro Pro Ser Thr 675 680 685 Ser Trp Gln Pro Gln Ser His Pro ArgGln Gly His Gly Thr Ser Pro 690 695 700 Val Gly Asp His Gly Ser Leu ValArg Ile Pro Pro Pro Pro Ala His 705 710 715 720 Glu Arg Ser Leu Ser AlaTyr Gly Gly Gly Asn Arg Ala Ser Met Glu 725 730 735 Phe Leu Arg Gln GluAsp Thr Pro Gly Cys Arg Pro Pro Glu Gly Thr 740 745 750 Leu Arg Asp SerAsp Thr Ser Ile Ser Ile Pro Ser Val Asp His Glu 755 760 765 Glu Leu GluArg Ser Phe Ser Gly Phe Ser Ile Ser Gln Ser Lys Glu 770 775 780 Asn LeuAsp Ala Leu Asn Ser Cys Tyr Ala Ala Val Ala Pro Cys Ala 785 790 795 800Lys Val Arg Pro Tyr Ile Ala Glu Gly Glu Ser Asp Thr Asp Ser Asp 805 810815 Leu Cys Thr Pro Cys Gly Pro Pro Pro Arg Ser Ala Thr Gly Glu Gly 820825 830 Pro Phe Gly Asp Val Gly Trp Ala Gly Pro Arg Lys 835 840 5 872PRT Homo sapiens 5 Met Gly Leu Lys Ala Arg Arg Ala Ala Gly Ala Ala GlyGly Gly Gly 1 5 10 15 Asp Gly Gly Gly Gly Gly Gly Gly Ala Ala Asn ProAla Gly Gly Asp 20 25 30 Ala Ala Ala Ala Gly Asp Glu Glu Arg Lys Val GlyLeu Ala Pro Gly 35 40 45 Asp Val Glu Gln Val Thr Leu Ala Leu Gly Ala GlyAla Asp Lys Asp 50 55 60 Gly Thr Leu Leu Leu Glu Gly Gly Gly Arg Asp GluGly Gln Arg Arg 65 70 75 80 Thr Pro Gln Gly Ile Gly Leu Leu Ala Lys ThrPro Leu Ser Arg Pro 85 90 95 Val Lys Arg Asn Asn Ala Lys Tyr Arg Arg IleGln Thr Leu Ile Tyr 100 105 110 Asp Ala Leu Glu Arg Pro Arg Gly Trp AlaLeu Leu Tyr His Ala Leu 115 120 125 Val Phe Leu Ile Val Leu Gly Cys LeuIle Leu Ala Val Leu Thr Thr 130 135 140 Phe Lys Glu Tyr Glu Thr Val SerGly Asp Trp Leu Leu Leu Leu Glu 145 150 155 160 Thr Phe Ala Ile Phe IlePhe Gly Ala Glu Phe Ala Leu Arg Ile Trp 165 170 175 Ala Ala Gly Cys CysCys Arg Tyr Lys Gly Trp Arg Gly Arg Leu Lys 180 185 190 Phe Ala Arg LysPro Leu Cys Met Leu Asp Ile Phe Val Leu Ile Ala 195 200 205 Ser Val ProVal Val Ala Val Gly Asn Gln Gly Asn Val Leu Ala Thr 210 215 220 Ser LeuArg Ser Leu Arg Phe Leu Gln Ile Leu Arg Met Leu Arg Met 225 230 235 240Asp Arg Arg Gly Gly Thr Trp Lys Leu Leu Gly Ser Ala Ile Cys Ala 245 250255 His Ser Lys Glu Leu Ile Thr Ala Trp Tyr Ile Gly Phe Leu Thr Leu 260265 270 Ile Leu Ser Ser Phe Leu Val Tyr Leu Val Glu Lys Asp Val Pro Glu275 280 285 Val Asp Ala Gln Gly Glu Glu Met Lys Glu Glu Phe Glu Thr TyrAla 290 295 300 Asp Ala Leu Trp Trp Gly Leu Ile Thr Leu Ala Thr Ile GlyTyr Gly 305 310 315 320 Asp Lys Thr Pro Lys Thr Trp Glu Gly Arg Leu IleAla Ala Thr Phe 325 330 335 Ser Leu Ile Gly Val Ser Phe Phe Ala Leu ProAla Gly Ile Leu Gly 340 345 350 Ser Gly Leu Ala Leu Lys Val Gln Glu GlnHis Arg Gln Lys His Phe 355 360 365 Glu Lys Arg Arg Lys Pro Ala Ala GluLeu Ile Gln Ala Ala Trp Arg 370 375 380 Tyr Tyr Ala Thr Asn Pro Asn ArgIle Asp Leu Val Ala Thr Trp Arg 385 390 395 400 Phe Tyr Glu Ser Val ValSer Phe Pro Phe Phe Arg Lys Glu Gln Leu 405 410 415 Glu Ala Ala Ser SerGln Lys Leu Gly Leu Leu Asp Arg Val Arg Leu 420 425 430 Ser Asn Pro ArgGly Ser Asn Thr Lys Gly Lys Leu Phe Thr Pro Leu 435 440 445 Asn Val AspAla Ile Glu Glu Ser Pro Ser Lys Glu Pro Lys Pro Val 450 455 460 Gly LeuAsn Asn Lys Glu Arg Phe Arg Thr Ala Phe Arg Met Lys Ala 465 470 475 480Tyr Ala Phe Trp Gln Ser Ser Glu Asp Ala Gly Thr Gly Asp Pro Met 485 490495 Ala Glu Asp Arg Gly Tyr Gly Asn Asp Phe Pro Ile Glu Asp Met Ile 500505 510 Pro Thr Leu Lys Ala Ala Ile Arg Ala Val Arg Ile Leu Gln Phe Arg515 520 525 Leu Tyr Lys Lys Lys Phe Lys Glu Thr Leu Arg Pro Tyr Asp ValLys 530 535 540 Asp Val Ile Glu Gln Tyr Ser Ala Gly His Leu Asp Met LeuSer Arg 545 550 555 560 Ile Lys Tyr Leu Gln Thr Arg Ile Asp Met Ile PheThr Pro Gly Pro 565 570 575 Pro Ser Thr Pro Lys His Lys Lys Ser Gln LysGly Ser Ala Phe Thr 580 585 590 Phe Pro Ser Gln Gln Ser Pro Arg Asn GluPro Tyr Val Ala Arg Pro 595 600 605 Ser Thr Ser Glu Ile Glu Asp Gln SerMet Met Gly Lys Phe Val Lys 610 615 620 Val Glu Arg Gln Val Gln Asp MetGly Lys Lys Leu Asp Phe Leu Val 625 630 635 640 Asp Met His Met Gln HisMet Glu Arg Leu Gln Val Gln Val Thr Glu 645 650 655 Tyr Tyr Pro Thr LysGly Thr Ser Ser Pro Ala Glu Ala Glu Lys Lys 660 665 670 Glu Asp Asn ArgTyr Ser Asp Leu Lys Thr Ile Ile Cys Asn Tyr Ser 675 680 685 Glu Thr GlyPro Pro Glu Pro Pro Tyr Ser Phe His Gln Val Thr Ile 690 695 700 Asp LysVal Ser Pro Tyr Gly Phe Phe Ala His Asp Pro Val Asn Leu 705 710 715 720Pro Arg Gly Gly Pro Ser Ser Gly Lys Val Gln Ala Thr Pro Pro Ser 725 730735 Ser Ala Thr Thr Tyr Val Glu Arg Pro Thr Val Leu Pro Ile Leu Thr 740745 750 Leu Leu Asp Ser Arg Val Ser Cys His Ser Gln Ala Asp Leu Gln Gly755 760 765 Pro Tyr Ser Asp Arg Ile Ser Pro Arg Gln Arg Arg Ser Ile ThrArg 770 775 780 Asp Ser Asp Thr Pro Leu Ser Leu Met Ser Val Asn His GluGlu Leu 785 790 795 800 Glu Arg Ser Pro Ser Gly Phe Ser Ile Ser Gln AspArg Asp Asp Tyr 805 810 815 Val Phe Gly Pro Asn Gly Gly Ser Ser Trp MetArg Glu Lys Arg Tyr 820 825 830 Leu Ala Glu Gly Glu Thr Asp Thr Asp ThrAsp Pro Phe Thr Pro Ser 835 840 845 Gly Ser Met Pro Leu Ser Ser Thr GlyAsp Gly Ile Ser Asp Ser Val 850 855 860 Trp Thr Pro Ser Asn Lys Pro Ile865 870 6 695 PRT Homo sapiens 6 Met Ala Glu Ala Pro Pro Arg Arg Leu GlyLeu Gly Pro Pro Pro Gly 1 5 10 15 Asp Ala Pro Arg Ala Glu Leu Val AlaLeu Thr Ala Val Gln Ser Glu 20 25 30 Gln Gly Glu Ala Gly Gly Gly Gly SerPro Arg Arg Leu Gly Leu Leu 35 40 45 Gly Ser Pro Leu Pro Pro Gly Ala ProLeu Pro Gly Pro Gly Ser Gly 50 55 60 Ser Gly Ser Ala Cys Gly Gln Arg SerSer Ala Ala His Lys Arg Tyr 65 70 75 80 Arg Arg Leu Gln Asn Trp Val TyrAsn Val Leu Glu Arg Pro Arg Gly 85 90 95 Trp Ala Phe Val Tyr His Val PheIle Phe Leu Leu Val Phe Ser Cys 100 105 110 Leu Val Leu Ser Val Leu SerThr Ile Gln Glu His Gln Glu Leu Ala 115 120 125 Asn Glu Cys Leu Leu IleLeu Glu Phe Val Met Ile Val Val Phe Gly 130 135 140 Leu Glu Tyr Ile ValArg Val Trp Ser Ala Gly Cys Cys Cys Arg Tyr 145 150 155 160 Arg Gly TrpGln Gly Arg Phe Arg Phe Ala Arg Lys Pro Phe Cys Val 165 170 175 Ile AspPhe Ile Val Phe Val Ala Ser Val Ala Val Ile Ala Ala Gly 180 185 190 ThrGln Gly Asn Ile Phe Ala Thr Ser Ala Leu Arg Ser Met Arg Phe 195 200 205Leu Gln Ile Leu Arg Met Val Arg Met Asp Arg Arg Gly Gly Thr Trp 210 215220 Lys Leu Leu Gly Ser Val Val Tyr Ala His Ser Lys Glu Leu Ile Thr 225230 235 240 Ala Trp Tyr Ile Gly Phe Leu Val Leu Ile Phe Ala Ser Phe LeuVal 245 250 255 Tyr Leu Ala Glu Lys Asp Ala Asn Ser Asp Phe Ser Ser TyrAla Asp 260 265 270 Ser Leu Trp Trp Gly Thr Ile Thr Leu Thr Thr Ile GlyTyr Gly Asp 275 280 285 Lys Thr Pro His Thr Trp Leu Gly Arg Val Leu AlaAla Gly Phe Ala 290 295 300 Leu Leu Gly Ile Ser Phe Phe Ala Leu Pro AlaGly Ile Leu Gly Ser 305 310 315 320 Gly Phe Ala Leu Lys Val Gln Glu GlnHis Arg Gln Lys His Phe Glu 325 330 335 Lys Arg Arg Met Pro Ala Ala AsnLeu Ile Gln Ala Ala Trp Arg Leu 340 345 350 Tyr Ser Thr Asp Met Ser ArgAla Tyr Leu Thr Ala Thr Trp Tyr Tyr 355 360 365 Tyr Asp Ser Ile Leu ProSer Phe Arg Glu Leu Ala Leu Leu Phe Glu 370 375 380 His Val Gln Arg AlaArg Asn Gly Gly Leu Arg Pro Leu Glu Val Arg 385 390 395 400 Arg Ala ProVal Pro Asp Gly Ala Pro Ser Arg Tyr Pro Pro Val Ala 405 410 415 Thr CysHis Arg Pro Gly Ser Thr Ser Phe Cys Pro Gly Glu Ser Ser 420 425 430 ArgMet Gly Ile Lys Asp Arg Ile Arg Met Gly Ser Ser Gln Arg Arg 435 440 445Thr Gly Pro Ser Lys Gln Gln Leu Ala Pro Pro Thr Met Pro Thr Ser 450 455460 Pro Ser Ser Glu Gln Val Gly Glu Ala Thr Ser Pro Thr Lys Val Gln 465470 475 480 Lys Ser Trp Ser Phe Asn Asp Arg Thr Arg Phe Arg Ala Ser LeuArg 485 490 495 Leu Lys Pro Arg Thr Ser Ala Glu Asp Ala Pro Ser Glu GluVal Ala 500 505 510 Glu Glu Lys Ser Tyr Gln Cys Glu Leu Thr Val Asp AspIle Met Pro 515 520 525 Ala Val Lys Thr Val Ile Arg Ser Ile Arg Ile LeuLys Phe Leu Val 530 535 540 Ala Lys Arg Lys Phe Lys Glu Thr Leu Arg ProTyr Asp Val Lys Asp 545 550 555 560 Val Ile Glu Gln Tyr Ser Ala Gly HisLeu Asp Met Leu Gly Arg Ile 565 570 575 Lys Ser Leu Gln Thr Arg Val AspGln Ile Val Gly Arg Gly Pro Gly 580 585 590 Asp Arg Lys Ala Arg Glu LysGly Asp Lys Gly Pro Ser Asp Ala Glu 595 600 605 Val Val Asp Glu Ile SerMet Met Gly Arg Val Val Lys Val Glu Lys 610 615 620 Gln Val Gln Ser IleGlu His Lys Leu Asp Leu Leu Leu Gly Phe Tyr 625 630 635 640 Ser Arg CysLeu Arg Ser Gly Thr Ser Ala Ser Leu Gly Ala Val Gln 645 650 655 Val ProLeu Phe Asp Pro Asp Ile Thr Ser Asp Tyr His Ser Pro Val 660 665 670 AspHis Glu Asp Ile Ser Val Ser Ala Gln Thr Leu Ser Ile Ser Arg 675 680 685Ser Val Ser Thr Asn Met Asp 690 695 7 854 PRT Homo sapiens 7 Met Lys AspVal Glu Ser Gly Arg Gly Arg Val Leu Leu Asn Ser Ala 1 5 10 15 Ala AlaArg Gly Asp Gly Leu Leu Leu Leu Gly Thr Arg Ala Ala Thr 20 25 30 Leu GlyGly Gly Gly Gly Gly Leu Arg Glu Ser Arg Arg Gly Lys Gln 35 40 45 Gly AlaArg Met Ser Leu Leu Gly Lys Pro Leu Ser Tyr Thr Ser Ser 50 55 60 Gln SerCys Arg Arg Asn Val Lys Tyr Arg Arg Val Gln Asn Tyr Leu 65 70 75 80 TyrAsn Val Leu Glu Arg Pro Arg Gly Trp Ala Phe Ile Tyr His Ala 85 90 95 PheVal Phe Leu Leu Val Phe Gly Cys Leu Ile Leu Ser Val Phe Ser 100 105 110Thr Ile Pro Glu His Thr Lys Leu Ala Ser Ser Cys Leu Leu Ile Leu 115 120125 Glu Phe Val Met Ile Val Val Phe Gly Leu Glu Phe Ile Ile Arg Ile 130135 140 Trp Ser Ala Gly Cys Cys Cys Arg Tyr Arg Gly Trp Gln Gly Arg Leu145 150 155 160 Arg Phe Ala Arg Lys Pro Phe Cys Val Ile Asp Thr Ile ValLeu Ile 165 170 175 Ala Ser Ile Ala Val Val Ser Ala Lys Thr Gln Gly AsnIle Phe Ala 180 185 190 Thr Ser Ala Leu Arg Ser Leu Arg Phe Leu Gln IleLeu Arg Met Val 195 200 205 Arg Met Asp Arg Arg Gly Gly Thr Trp Lys LeuLeu Gly Ser Val Val 210 215 220 Tyr Ala His Ser Lys Glu Leu Ile Thr AlaTrp Tyr Ile Gly Phe Leu 225 230 235 240 Val Leu Ile Phe Ser Ser Phe LeuVal Tyr Leu Val Glu Lys Asp Ala 245 250 255 Asn Lys Glu Phe Ser Thr TyrAla Asp Ala Leu Trp Trp Gly Thr Ile 260 265 270 Thr Leu Thr Thr Ile GlyTyr Gly Asp Lys Thr Pro Leu Thr Trp Leu 275 280 285 Gly Arg Leu Leu SerAla Gly Phe Ala Leu Leu Gly Ile Ser Phe Phe 290 295 300 Ala Leu Pro AlaGly Ile Leu Gly Ser Gly Phe Ala Leu Lys Val Gln 305 310 315 320 Glu GlnHis Arg Gln Lys His Phe Glu Lys Arg Arg Asn Pro Ala Ala 325 330 335 AsnLeu Ile Gln Cys Val Trp Arg Ser Tyr Ala Ala Asp Glu Lys Ser 340 345 350Val Ser Ile Ala Thr Trp Lys Pro His Leu Lys Ala Leu His Thr Cys 355 360365 Ser Pro Thr Lys Lys Glu Gln Gly Glu Ala Ser Ser Ser Gln Lys Leu 370375 380 Ser Phe Lys Glu Arg Val Arg Met Ala Ser Pro Arg Gly Gln Ser Ile385 390 395 400 Lys Ser Arg Gln Ala Ser Val Gly Asp Arg Arg Ser Pro SerThr Asp 405 410 415 Ile Thr Ala Glu Gly Ser Pro Thr Lys Val Gln Lys SerTrp Ser Phe 420 425 430 Asn Asp Arg Thr Arg Phe Arg Pro Ser Leu Arg LeuLys Ser Ser Gln 435 440 445 Pro Lys Pro Val Ile Asp Ala Asp Thr Ala LeuGly Thr Asp Asp Val 450 455 460 Tyr Asp Glu Lys Gly Cys Gln Cys Asp ValSer Val Glu Asp Leu Thr 465 470 475 480 Pro Pro Leu Lys Thr Val Ile ArgAla Ile Arg Ile Met Lys Phe His 485 490 495 Val Ala Lys Arg Lys Phe LysGlu Thr Leu Arg Pro Tyr Asp Val Lys 500 505 510 Asp Val Ile Glu Gln TyrSer Ala Gly His Leu Asp Met Leu Cys Arg 515 520 525 Ile Lys Ser Leu GlnThr Arg Val Asp Gln Ile Leu Gly Lys Gly Gln 530 535 540 Ile Thr Ser AspLys Lys Ser Arg Glu Lys Ile Thr Ala Glu His Glu 545 550 555 560 Thr ThrAsp Asp Leu Ser Met Leu Gly Arg Val Val Lys Val Glu Lys 565 570 575 GlnVal Gln Ser Ile Glu Ser Lys Leu Asp Cys Leu Leu Asp Ile Tyr 580 585 590Gln Gln Val Leu Arg Lys Gly Ser Ala Ser Ala Leu Ala Leu Ala Ser 595 600605 Phe Gln Ile Pro Pro Phe Glu Cys Glu Gln Thr Ser Asp Tyr Gln Ser 610615 620 Pro Val Asp Ser Lys Asp Leu Ser Gly Ser Ala Gln Asn Ser Gly Cys625 630 635 640 Leu Ser Arg Ser Thr Ser Ala Asn Ile Ser Arg Gly Leu GlnPhe Ile 645 650 655 Leu Thr Pro Asn Glu Phe Ser Ala Gln Thr Phe Tyr AlaLeu Ser Pro 660 665 670 Thr Met His Ser Gln Ala Thr Gln Val Pro Ile SerGln Ser Asp Gly 675 680 685 Ser Ala Val Ala Ala Thr Asn Thr Ile Ala AsnGln Ile Asn Thr Ala 690 695 700 Pro Lys Pro Ala Ala Pro Thr Thr Leu GlnIle Pro Pro Pro Leu Pro 705 710 715 720 Ala Ile Lys His Leu Pro Arg ProGlu Thr Leu His Pro Asn Pro Ala 725 730 735 Gly Leu Gln Glu Ser Ile SerAsp Val Thr Thr Cys Leu Val Ala Ser 740 745 750 Lys Glu Asn Val Gln ValAla Gln Ser Asn Leu Thr Lys Asp Arg Ser 755 760 765 Met Arg Lys Ser PheAsp Met Gly Gly Glu Thr Leu Leu Ser Val Cys 770 775 780 Pro Met Val ProLys Asp Leu Gly Lys Ser Leu Ser Val Gln Asn Leu 785 790 795 800 Ile ArgSer Thr Glu Glu Leu Asn Ile Gln Leu Ser Gly Ser Glu Ser 805 810 815 SerGly Ser Arg Gly Ser Gln Asp Phe Tyr Pro Lys Trp Arg Glu Ser 820 825 830Lys Leu Phe Ile Thr Asp Glu Glu Val Gly Pro Glu Glu Thr Glu Thr 835 840845 Asp Thr Phe Ala Arg Ile 850 8 29 PRT Homo sapiens 8 Lys Lys Glu GlnGly Glu Ala Ser Ser Asn Lys Phe Cys Ser Asn Lys 1 5 10 15 Gln Lys LeuPhe Arg Met Tyr Thr Ser Arg Lys Gln Ser 20 25 9 9 PRT Homo sapiens 9 LysLys Glu Gln Gly Glu Ala Ser Ser 1 5 10 20 PRT Homo sapiens 10 Asn LysPhe Cys Ser Asn Lys Gln Lys Leu Phe Arg Met Tyr Thr Ser 1 5 10 15 ArgLys Gln Ser 20

1. An isolated nucleic acid molecule encoding a polypeptide comprisingall or a portion of a human, rat or murine KCNQ5 protein.
 2. An isolatedpolynucleotide having a nucleic acid sequence which is capable ofhybridizing under at least medium stringency conditions with thepolynucleotide sequence presented as SEQ ID NO: 1, its complementarystrand, or a subsequence thereof.
 3. The isolated polynucleotideaccording to claim 2, being at least 65% homologous, preferably morethan 70%, more preferred more than 80%, even more preferred more than90%, most preferred more than 95%, homologous to the polynucleotidesequence presented as SEQ ID NO:
 1. 4. The isolated polynucleotideaccording to any of claims 1-3, wherein said polynucleotide is a clonedpolynucleotide.
 5. The isolated polynucleotide according to claim 4,wherein the polynucleotide is cloned from, or produced on the basis of acDNA library.
 6. The isolated polynucleotide according to claim 1,comprising the polynucleotide sequence presented as SEQ ID NO:
 1. 7. Theisolated polynucleotide according to claim 1, encoding a potassiumchannel, or a potassium channel subunit.
 8. The isolated polynucleotideaccording to claim 7, encoding the KCNQ5 potassium channel subunitcomprising the amino acid sequence represented by SEQ ID NO:
 2. 9. Theisolated polynucleotide according to claim 7, encoding a KCNQ5 variant,which variant has an amino acid sequence that has been changed bydeletion of an amino acid residue, by insertion of an additional aminoacid residue, or by substitution of an amino acid residue at one or morepositions.
 10. The isolated polynucleotide according to claim 9, whereinsaid variant has an amino acid sequence that has been changed at one ormore positions located in the conserved regions, as defined by Table 1.11. The isolated polynucleotide according to claim 9, wherein saidvariant is G329S (KCNQ1 numbering), or KCNQ5/G278S.
 12. A vectorconstruct comprising the polynucleotide according to claim
 11. 13. Arecombinantly produced polypeptide encoded by the polynucleotideaccording to claim
 1. 14. The polypeptide according to claim 13, whereinsaid polypeptide is a KCNQ5 potassium channel subunit having the aminoacid sequence presented as SEQ ID No.
 2. 15. The polypeptide of eitherof claims 13-14, comprising a molecular weight of approximately 99 kDa.16. The polypeptide of claim 13, comprising six transmembrane domains, aP-loop, and a carboxy-terminal conserved cytoplasmic region (the“A-domain”).
 17. The polypeptide according to claim 13, wherein saidpolypeptide is a KCNQ5 variant, which variant has an amino acid sequencethat has been changed by deletion of an amino acid residue, by insertionof an additional amino acid residue, or by substitution of an amino acidresidue at one or more positions.
 18. The polypeptide according to claim17, wherein said variant has an amino acid sequence that has beenchanged at one or more positions located in the conserved regions, asdefined by Table
 1. 19. The polypeptide according to claim 18, whereinsaid variant is G329S (KCNQ1 numbering), or KCNQ5/G278S.
 20. A cellgenetically manipulated by the incorporation of a heterologouspolynucleotide according to claim 1 or a vector construct according toclaim
 12. 21. The cell according to claim 20, genetically manipulated bythe incorporation of a KCNQ5 channel subunit having the amino acidsequence presented as SEQ ID NO:
 2. 22. The cell according to claim 20,genetically manipulated by the incorporation of a KCNQ5 variant, whereinsaid variant has an amino acid sequence that has been changed bydeletion of an amino acid residue, by insertion of an additional aminoacid residue, or by substitution of an amino acid residue at one or morepositions.
 23. The cell according to claim 22, wherein said variant hasan amino acid sequence that has been changed at one or more positionslocated in the conserved regions, as defined by Table
 1. 24. The cellaccording to claim 20, genetically manipulated to co-express one or moreKCNQ channel subunits.
 25. The cell according to claim 24, geneticallymanipulated to co-express KCNQ5 and KCNQ1 channel subunits; KCNQ5 andKCNQ2 channel subunits; KCNQ5 and KCNQ3 channel subunits; KCNQ5 andKCNQ4 channel subunits; KCNQ5 and KCNQ1 and KCNQ2 channel subunits;KCNQ5 and KCNQ1 and KCNQ3 channel subunits; KCNQ5 and KCNQ1 and KCNQ4channel subunits; KCNQ5 and KCNQ2 and KCNQ3 channel subunits; KCNQ5 andKCNQ2 and KCNQ4 channel subunits; KCNQ5 and KCNQ3 and KCNQ4 channelsubunits; KCNQ5 and KCNQ1 and KCNQ2 and KCNQ3 channel subunits; KCNQ5and KCNQ1 and KCNQ2 and KCNQ4 channel subunits; KCNQ5 and KCNQ1 andKCNQ3 and KCNQ4 channel subunits; or KCNQ5 and KCNQ2 and KCNQ3 and KCNQ4channel subunits.
 26. The cell according to claim 24, geneticallymanipulated to co-express KCNQ2 or KCNQ3, and KCNQ5 channel subunits.27. The cell according to claim 20, wherein said cell is an eukaryoticcell, in particular a mammalian cell, an oocyte, or a yeast cell. 28.The cell according to any claim 27, being a human embryonic kidney (HEK)cell, a HEK 293 cell, a BHK21 cell, a Chinese hamster ovary (CHO) cell,a Xenopus laevis oocyte (XLO) cell, a COS cell, or any other cell lineable to express KCNQ potassium channels.
 29. A membrane preparationderived from a cell according to claim
 20. 30. A method for obtaining asubstantially homogeneous source of a human potassium channel,comprising a KCNQ5 subunit, which method comprises the steps ofculturing a cellular host having incorporated expressibly therein apolynucleotide according to claim 1, or a vector construct according toclaim 12, and then recovering the cultured cells.
 31. The method ofclaim 29, further comprising the subsequent step of obtaining a membranepreparation from the cultured cells.
 32. A method of screening achemical compound for capability of binding to a potassium channelcomprising at least one KCNQ5 channel subunit, which method comprisesthe steps of (i) subjecting a KCNQ5 channel subunit containing cell, ora membrane preparation hereof, to the action of a KCNQ5 binding agent toform a complex with the KCNQ5 channel subunit containing cell; (ii)subjecting the complex of step (i) to the action of the chemicalcompound to be tested; and (iii) detecting the displacement of the KCNQ5binding agent from the complex with the KCNQ5 channel subunit containingcell.
 33. The method of claim 32, wherein the KCNQ5 channel subunitcontaining cell is a cell according to claim 20, or a membranepreparation according to claim
 29. 34. The method of claim 32, whereinthe KCNQ5 binding agent is (i) radioactively labeled1,3-dihydro-1-phenyl-3,3-bis-(4-pyridylmethyl)-2H-indol-2-one(Linopirdine);or (ii) radioactively labeled 10,10-bis-(4-pyridinyl-methyl)-9-(1OH)-anthra-cenone(XE991).
 35. The method of claim 34, wherein saidcompounds have been marked with ³H.
 36. The method of either of claims32-33, wherein the displacement of the KCNQ5 binding agent from thecomplex with the KCNQ5 channel subunit containing cell is detected bymeasuring the amount of radioactivity by conventional liquidscintillation counting.
 37. A method of screening a chemical compoundfor activity on a potassium channel comprising at least one KCNQ5channel subunit, which method comprises the steps of (i) subjecting aKCNQ5 channel subunit containing cell, or a membrane preparation hereof,to the action of the chemical compound; and (ii) monitoring the membranepotential, the current, the potassium flux, or the secondary calciuminflux of the KCNQ5 channel subunit containing cell.
 38. The method ofclaim 37, wherein the KCNQ5 channel subunit containing cell is a cellaccording to claim 20, or a membrane preparation according to claim 29.39. The method of claim 37, wherein monitoring of the membrane potentialof the KCNQ5 channel subunit containing cell is performed by patch clamptechniques.
 40. The method of claim 37, wherein monitoring of themembrane potential of the KCNQ5 channel subunit containing cell isperformed using fluorescence methods.
 41. A chemical compound identifiedby the method of claim 32 and/or claim
 37. 42. A method for diagnosis,treatment, prevention or alleviation of diseases related to diseases oradverse conditions of the CNS, including affective disorders,Alzheimer's disease, anxiety, ataxia, CNS damage caused by trauma,stroke or neurodegenerative illness, cognitive deficits, compulsivebehavior, dementia, depression, Huntington's disease, mania, memoryimpairment, memory disorders, memory dysfunction, motion, disorders,motor disorders, neurodegenerative diseases, Parkinson's disease andParkinson-like motor disorders, phobias, Pick's disease, psychosis,schizophrenia, spinal cord damage, stroke, tremor, seizures, convulsionsand epilepsy, comprising the step of administering the chemical compoundof claim 41 to a subject in need thereof.
 43. The method according toclaim 42, wherein the chemical compound is1,3-dihydro-1-phenyl-3,3-bis-(4-pyridylmethyl)-2H-indol-2-one(Linopirdine);or 10,10-bis-(4-pyridinyl-methyl)-9-(1 OH)-antracenone(XE991).
 44. Amethod for determining individuals having mutations in thepolynucleotide sequence according to claim 1, comprising the step ofscreening of genetic materials collected from mammalian tissues, inparticular human tissues with the polynucleotide sequence according toclaim 1, or a vector construct according to claim
 12. 45. A transgenicanimal comprising a knock-out mutation of the endogenous KCNQ5 gene, amutated KCNQ5 gene, or genetically manipulated in order to over-expressthe KCNQ5 gene or to over-express mutated KCNQ5 gene.
 46. The transgenicanimal according to claim 45, wherein said animal is a knock-out animalin which the gene is totally deleted in a homozygous state.
 47. Thetransgenic animal according to claim 45, comprising a mutated KCNQ5gene.
 48. The transgenic animal according to claim 45, wherein saidanimal is a transgenic rodent, in particular a hamster, a guinea pig, arabbit, or a rat, a transgenic pig, a transgenic cattle, a transgenicsheep, or a transgenic goat.
 49. A method for in vivo screening oftherapeutic compounds, comprising the step of administering saidcompounds to the transgenic animal according to claim
 45. 50. The methodaccording to claim 49, for the screening of drugs affecting diseases orconditions associated with malfunction of the CNS, such as affectivedisorders, Alzheimer's disease, anxiety, ataxia, CNS damage caused bytrauma, stroke or neurodegenerative illness, cognitive deficits,compulsive behavior, dementia, depression, Huntington's disease, mania,memory impairment, memory disorders, memory dysfunction, motiondisorders, motor disorders, neurodegenerative diseases, Parkinson'sdisease and Parkinson-like motor disorders, phobias, Pick's disease,psychosis, schizophrenia, spinal cord damage, stroke, tremor, seizures,convulsions and epilepsy.
 51. An antibody capable of binding one or morepolypeptides as claimed in claim
 13. 52. The antibody of claim 51,wherein said antibody is a monoclonal antibody.